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r853 
.U5601 

1965 



mhesda, Maryland 2OOI4 



NATIONAL INSTITUTES OF HEALTH 



Review 
of 






INTRAMURAL 

RESEARCH 

1965 



N 




U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 



National Institutes ot HeaJW 
Bathesda, Maryland MU 



ISSUED IN A LIMITED EDITION FOR ADMINISTRATIVE USE 



NATIONAL INSTITUTES OF HEALTH 



Revi 



eview 



of 

INTRAMURAL 

RESEARCH 

1965 




U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

PubKc Health Service 
National Institutes of Health, Bethesda, Maryland 20014 



INSTITUTE RESEARCH DIRECTORS 

National Cancer Institute Charles G. Zubrod, M.D. 

Scientific Director 

Nathaniel I. Berlin, M.D., Ph.D. 

Clinical Director 

National Heart Institute Robert W. Berliner, M.D. 

Scientific Director 

Donald S. Fredrickson, M.D. 

Clinical Director 

National Institute of Arthritis and Metabolic Diseases Joseph E. Rail, M.D., Ph.D. 

Scientific Director 

Robert S. Gordon, Jr., M.D. 

Clinical Director 

National Institute of Allergy and Infectious Diseases Leon Jacobs, Ph.D. 

Acting Scientific Director 
Vernon Knight, M.D. 

Clinical Director 

National Institute of Mental Health John C. Eberhart, Ph.D. 

Scientific Director 
Robert A. Cohen, M.D., Ph.D. 

Clinical Director 

National Institute of Neurological Diseases and Blindness Karl Frank, Ph.D. 

Acting Scientific Director 
Maitland Baldwin, M.D. 

Clinical Director 

National Institute of Dental Research Seymour Kreshover, M.D., D.D.S., Ph.D. 

Scientific Director 
Edward J. DriscoU, D.D.S 

Clinical Director 

Division of Biologics Standards Roderick Murray, M.D. 

Scientific Director 



G. Burroughs Mider, M.D. 
Director of Laboratories and Clinics 

Willlam C. Mohler, M.D. 
Assistant to Director of Laboratories and Clinics 

III 



FOREWORD 

Here is the fifth review of Intramural Research from the National Institutes of Health. 
As the title implies the volume summarizes work done by the scientists in our own labora- 
tories and clinics and in the offices connected with these. It does not deal with the many 
extramural activities of the National Institutes of Health that support much of the 
biomedical research throughout the United States and some efforts in other nations. 

This review covers the year July 1964 through June 1965. There was no volume for the 
period in January 1963 through June 1964 when we changed from a calendar year to the 
present fiscal year basis. The philosophy behind the review has not changed. The 
words are those of the scientists, who followed very general guidelines. The reports 
received only minimal editing. Thus, we have tried to present for the reader as direct a 
view as possible into the workings of our large, diverse and very active scientific com- 
munity as it pursues the specific research goals given by Congress to the individual In- 
stitutes, which together comprise the principal research arm of the United States 
Public Health Service. The Institutes strive to extend fundamental knowledge about the 
health problems of man and thus to provide a base for the broader statutory concerns 
of the whole Public Health Service, namely, "the causes, diagnosis, treatment, control 
and prevention of physical and mental diseases and impairments of man." 



James A. Shannon, M.D. 



CONTENTS 

Page 

Foreword v 

NATIONAL CANCER INSTITUTE 1 

Laboratory of Biochemistry 1 

Cytochemistry Section 1 

Nucleic Acids Section 3 

Nutrition and Carcinogenesis Section 5 

Protein Chemistry Section 7 

Tumor-Host Relations Section 11 

Laboratory of Biology 14 

Immunology 15 

Virology 15 

Biochemistry 16 

Mammalian Genetics 17 

Endocrinology 18 

Cell Physiology and Nutrition 18 

Tissue Culture Methodology 19 

Pathologic Anatomy Branch 19 

Laboratory of Pathology 20 

Cancer in Man 20 

Cancer in Animals 23 

Viral Carcinogenesis 24 

Accumulation of Data . 25 

Collaborative Research 26 

Laboratory of Physiology 27 

Office of the Chief 27 

Cancer Physiology Section 28 

Energy Metabolism Section 29 

Physical Biology Section 29 

Radiation Biology Section 30 

Laboratory of Chemical Pharmacology 31 

Medicine Branch 34 

Lymphomas 34 

Combination Therapy in Solid Tumors 35 

Chronic Myelocytic Leukemia 35 

Acute Leukemia 35 

Multiple Myeloma 35 

New Agents 36 

Leukocyte Dynamics in Leukemia 36 

Leukocyte and Platelet Transfusion 36 

Life Island 37 



vn 



VIU CONTENTS 

Page 

Leukemic Leukocytes and Malignant Cells 37 

Virus Studies 38 

Laboratories of Viral Carcinogenesis and Viral Oncology 38 

Clinical Investigations 48 

Amino Acid Metabolism 48 

Calcium Metabolism 50 

Nucleic Acid and Pyrimidine Metabolism 50 

Metabolism of Plasma Proteins 51 

Porphyrin Metabolism 53 

Erythropoiesis 54 

Bilirubin Turnover 54 

Dermatology Branch 55 

Mycosis Fungoides Lymphoma 55 

Epidermal Growth and Differentiation 56 

Pteridine Metabolism and Pigmentation 56 

Diagnostic Research Branch 56 

Office of the Chief 56 

Endocrinology Branch 57 

Immunology Branch 58 

Radiation Branch 61 

Surgery Branch 62 

NATIONAL HEART INSTITUTE 69 

Laboratory of Biochemistry 69 

Section on Enzymes 69 

Divergent Biosynthetic Pathways of Metabolism 69 

One Carbon Metabolism 70 

Metabolism of Amino Acids 70 

Cystathionine Biosynthesis and Trans-Sulfuration 71 

Action of Vitamin B12 Coenzyme 72 

Anaerobic Hydroxylation of Aromatic Compounds 73 

Uptake of Amino Acids 73 

Metabolism of Heterocyclic Compounds 74 

Section on Comparative Biochemistry 74 

Structure and Substrate Binding Site of ACP 75 

ACP Involvement in Lipid Metabolism 75 

ACP Involvement in Fatty Acid Biosynthesis 77 

Biosynthesis of ACP 78 

ACP in Mammalian Tissues 78 

Section on Cellular Physiology 79 

Myosin 79 

Bovine Fibrinogen 79 

Ribonuclease 80 

Protein Biosynthesis 80 

Nonphosphorylated High-Energy Intermediates 80 

Synthesis of Unsaturated Fatty Acids 81 

Cell Transport 81 

Laboratory of Chemical Pharmacology 82 

Biological Control Systems 82 

Adrenergical Neurochemical Transducer 82 



CONTENTS IX 

Page 

Release of NE by Various Drugs 82 

NE release at Molecular and Subcellular Levels 83 

Effects of DMI 83 

Recapture Process 84 

Rates of NE Synthesis 84 

Other Research 85 

Serotonergic Transducer 86 

Effects of Drugs in Various Phyla 86 

Nonmast Cell Histamine 87 

Sympathetic Target Sites 88 

Adipose Tissue System 88 

Sympathetic and Thyroid Systems 89 

Nicotinic-acid Induced Inhibition of Lipolysis 89 

Alpha and Beta Adrenergic Blockade 89 

Lipolji;ic Effects on Theophylline 89 

Biochemical Lesions in Adrenalectomy 89 

Factors Which Affect Drug Action 90 

Thalidomide 90 

Desmethylimipramine 91 

Enzymatic Mechanism of Drug Metabolism 91 

Passage of Substances Across Membranes 91 

Central Nervous System 91 

Biliary Excretion of Drugs 92 

Enzymatic Membrane Transport 92 

Uptake and Storage of Catecholamines 92 

New Methods of Analysis 93 

Laboratory of Technical Development 93 

Fluorescence Methods 93 

Ultramicro Methods 94 

Artificial Organs 95 

Blood Oxygenator 95 

Blood Pumping 95 

Blood Dialyzer 96 

Fast Reaction Methods 96 

Gas Chromatography 97 

Blood Flowmetering 97 

Mathematics and Computers 98 

Laboratory of Cardiovascular Physiology 98 

Potassium Changes in the Heart 98 

Myocardial Oxj^gen Consumption 100 

Peripheral Vascular Resistance in Exercise 100 

Renal Studies 102 

Intravascular Receptors 102 

Kallikrein System 102 

Electrical Stimulation of the Heart 103 

Laboratory of Kidney and Electroljrte Metabolism 103 

Micropuncture Studies 103 

Isolated Tubules 104 

Toad Bladder 105 

Electrolyte Transport 107 



CONTENTS 

Page 

Renin-Angiotensin Aldosterone System 108 

Cardiac Muscle Contraction HO 

Laboratory of Metabolism HO 

Section on Metabolism HI 

Mobilization of Free Fatty Acids HI 

Refsum's Syndrome H3 

Lymphatic Lipids H4 

Membrane Lipoproteins H5 

Cholesterol Biosynthesis H5 

Section of Molecular Disease H5 

Protein Structure and Function 115 

Plasma Lipids and Lipoproteins 116 

Section on Chemistry 116 

Laboratory of Clinical Biochemistry 117 

Amine Biogenesis and Metabolism 117 

Collagen and Hydroxyproline 117 

Amino Acid Uptake 118 

Biosynthesis of Nitrogen Containing Lipids 118 

RNA Metabolism in Brain 118 

Proteins and Peptides 118 

Aromatic Hydroxylation and Metabolism 119 

Vitamin B12 and Methionine 119 

Section of Biochemical Genetics 119 

Clinical Endocrinology Branch 120 

Adrenal Cortical Steroids 120 

Renal Control of Salt and Water Metabolism 122 

Calcium and Phosphorus Metabolism 123 

Miscellaneous Studies 124 

Experimental Therapeutics Branch 125 

Vasoactive Amines 125 

Kinin Peptides 126 

Selected Proteins 126 

Cardiology Branch 127 

Myocardial Energy Exchange 128 

Myocardial Contraction 129 

Digitalis 134 

Circulatory Control by Autonomic Nervous System 136 

Regional Circulations 139 

Coronary Circulation 139 

Limb Circulation 139 

Pulmonary Circulation 140 

Cardiac Diagnostic Methods 140 

Clinical Studies 141 

Section of Clinical Biophysics 142 

Pulmonary Emphysema 143 

Myocardial Mechanics 144 

Vascular Mechanics 145 

Surgery Branch 146 



CONTENTS XI 

Page 

Gerontology Branch 149 

Aging in the Human 149 

Physiological Basis of Behavior 149 

Age Changes in Psychological Performance 150 

Pulmonary Physiology 150 

Renal Physiology 151 

Endocrinology 152 

Metabolism 153 

Biology of Aging 154 

Behavioral Changes with Age 154 

Physiological Systems — Heart 154 

Connective Tissue 155 

Nutrition and Aging 155 

"Error" Theory of Cellular Aging 156 

Age Pigment 157 

Cell Death 157 

Basic Biology 158 

Intermediary Metabolism 158 

Molecular Biology— DNA and RNA 159 

NATIONAL INSTITUTE OF ARTHRITIS AND 

METABOLIC DISEASES 161 

Mathematical Research 162 

Laboratory of Biophysical Chemistry 163 

Laboratory of Experimental Pathology 164 

Pathology of Rheumatic Disease 164 

Scientific Literature Retrieval 165 

Chromosome Studies in Chronic Myelogenous Leukemia 165 

Histochemical Enzymes 165 

Human Lymphocytes 166 

RNA Metabolism 167 

Nucleic Acid and Protein Synthesis 167 

Soluble Ribonucleic Acid (s-RNA) 167 

DNA in Crithidia 167 

Phosphatases oni5^. Coli 168 

Histidinol Phosphate Phosphatase 168 

Localization of Antibodies 168 

Fluorescein 168 

Carbohydrate Histochemistry 168 

Histochemistry of Mucopolysaccharides 169 

Electron Microscopy 170 

Epinephrine 170 

Erythropoietin Production 171 

Adrenalectomy 171 

Dipetalonema witei 171 

Toxic Compounds 172 

Cycasin Research 172 

Excretion of Ingested Cycasin 173 

Cycasin and Its Aglycone 173 



Xll CONTENTS 

Page 

Laboratory of Chemical Biology 174 

Structure-Function Relationships in Proteins 174 

Tertiary Structure of Proteins 174 

Protein Biosynthesis 175 

Proteins and Human Heredity Disease 176 

Laboratory of Biochemical Pharmacology 176 

Mammalian Enzymes 176 

Microbial Physiology 177 

Spermine, Spermidine, and Other Amines 177 

Experimental Burns 178 

Leprosy 178 

Sulfur-Containing Compounds 178 

Fasting 179 

Laboratory of Biochemistry and Metabolism 179 

Carbohydrate Metabolism 179 

Metabolism of Hydrogen 180 

Hormones 180 

Nucleic Acids 180 

Enzyme Induction 181 

Surface Enzymes 181 

Laboratory of Physical Biology 182 

Molecular Structure 182 

Metabolism 184 

Biological Energy Transduction 184 

Laboratory of Nutrition and Endocrinology 186 

Fat-Soluble Vitamins 186 

Protein Deprivation 186 

Lipid Metabolism and Purine-Pyrimidine Balance 187 

Folic Acid 188 

Experimental Nutrition 188 

Parathyroid Hormone 189 

Pituitary Hormones 190 

Metabolism of Adipose Tissue 191 

Experimental Diabetes 193 

Laboratory of Chemistry 194 

Section on Carbohydrates 194 

Synthesis of Nucleosides 194 

Synthesis of Starfish Sialic Acid 195 

Higher-Carbon Sugars 195 

Section on Medicinal Chemistry 196 

Analgesics 196 

Aldolase Inhibitors 196 

Immunochemistry 197 

Section on Metabolites 198 

Section on Analytical Services and Instrumentation 203 

Section on Steroids 203 

Laboratory of Molecular Biology 204 

Glutamate Dehydrogenase 204 

Enzyme Induction in Bacteria 205 



CONTENTS XHl 

Page 

Enzyme Induction in Mammalian Cells 205 

Serine Transacetylase 205 

Steroid Hormones 205 

Amino Acid Transport 205 

Amino Acid Incorporation into Protein 206 

Isoguanosine 206 

Optical Properties of Nucleic Acid 206 

Internal Proteins of Bacteriophage T2 207 

Viscosity Studies of DNA 207 

Structural Studies on Nucleic Acids 208 

Histidine Biosynthesis 209 

Hemoglobin 209 

Lambda Bacteriophage Induction 209 

Host-Induced Modification 209 

Clinical Investigations 210 

Arthritis and Rheumatism Branch 210 

Rheumatic Diseases 210 

Lymphoma and Sjogren's Syndrome 210 

Effects of AUopurionl on Gout 210 

Serology and Immunochemistry 211 

Gamma Globulin in Rubella 211 

Macroglobulin 212 

Immunoglobulin 212 

Biochemical Studies 212 

Protein Synthesis in Spleen 212 

Glutamic Dehydrogenase 212 

Biochemistry of ACTH 212 

Enzyme Induction 213 

Gastroenterology Unit 213 

Small Intestine 213 

Whipple's Intestinal Lipodystrophy 213 

Intestinal Mucosa 214 

Metabolic Errors — Homocystinuria 214 

Clinical Endocrinology Branch 215 

Thyroid Biochemistry 215 

Anterior Pituitary Hormones 218 

Growth Hormone 218 

Gonadotropins 219 

Thyrotropins 219 

Steroid Hormones 219 

Insulin, Glucagon, and Carbohydrate Metabolism 220 

Amino Acid Transport 221 

Protein Synthesis and Structure 221 

Peptide Hormones 221 

Clinical Hematology Branch 222 

Immunology of Blood Cell Deficiencies 222 

Plasma Factor in Thrombocytopenic Purpura 222 

Cellular Sequestration 222 

Reticuloendothelial Blockade 223 



Xiv CONTENTS 

Page 

Transplantation Immunity 223 

Immunologic Therapy of Leukemia 224 

Isoimmune Neonatal Purpura 225 

Blood Coagulation and Diseases of Hemorrhage and Thrombosis — 225 

Surgery in Hemophiliacs 225 

Aldrich Syndrome 226 

Thrombocytopenia 226 

Purpura 227 

Metabolic Diseases Branch 227 

Mineral Metabolism 227 

Bone Metabolism 227 

Connective Tissue 228 

Energy Metabolism 229 

Immobilization 229 

Temperature Regulation 230 

Alterations in Endocrine Function 231 

Electrolyte Balance in Heat Stress 232 

Pediatric Metabolism Branch 232 

Immunochemistry 232 

Effects of Diuretics on Sodium Transport 234 

Cystic Fibrosis 235 

NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS 

DISEASES 237 

Introduction 237 

Laboratory of Parasite Chemotherapy 238 

Malaria— Human 238 

Malaria — Simian 239 

Entomology 240 

Immunology 240 

Chemotherapy 240 

Biochemistry 240 

Laboratory of Tropical Virology 241 

Serology 241 

Tissue Culture 241 

Immunity 241 

Laboratory of Bacterial Diseases 241 

Mycoplasma 241 

Brucella 242 

Laboratory of Germ-Free Animal Research 242 

Autoallergic Disease 242 

Immunoglobulins 242 

Eaton Agent 243 

Monocontaminated Mice 243 

Macroglobuli Levels 243 

Autoallergic Thyroiditis 243 

Allergic Encephalomyelitis 244 

Thyroid Transplantation 244 

Laboratory of Immunology 244 

Homograft Tolerance 244 



CONTENTS XV 



Hypersensitivity 245 

ENA in Immune Response 245 

Enterobacterial Antigen 245 

Immunosuppression by Drugs 245 

Skin-Sensitizing Antibodies 246 

Gene Expression 246 

Gamma Globulin 246 

Laboratory of Clinical Investigations 246 

Adenovirus Groups and Vaccines 246 

Equine Influenza 247 

Respiratory Viral Disease 247 

Rhinovirus Common Cold 247 

Gamma-G Immunoglobulin 247 

Gene Transplantation 247 

Endotoxin Tolerance 248 

Fevers 248 

Fungal Disease 248 

Laboratory of Parasitic Diseases 248 

Vitamin Bi^ — Nematode Relations 248 

Schistosomiasis 249 

Angiostrongyliasis 249 

Filariasis 249 

Host Diet 250 

Amebiasis 250 

Physiologic and Biochemical Studies 250 

Energy Metabolism 251 

Malaria — Paralysis Factor 251 

Toxoplasmosis and Sarcosporidiosis 251 

Immunology 251 

Laboratory of Biology of Viruses 252 

Polynucleotide Homologies 252 

Viral Replication 252 

Interferon 252 

Viral Oncogenesis 253 

Genetics 253 

Laboratory of Infectious Diseases 253 

Enteric Capsule Vaccine 253 

Syncytial Virus Vaccine 253 

PAP Vaccine 253 

Cancer-Inducing Viruses 254 

Leukemia Viruses 255 

Perinatal Disease 255 

Eosinophilic Meningitis 255 

ECHO Virus 256 

Measles Vaccine 256 

Autotrophic Hydrogenomonad 256 

Staphylococcal Cell Walls 256 

Bacterial Pathogens 256 

Pathogenic Fungi 257 



XVI CONTENTS 

Page 

Antifungal Agents 257 

Mycotic Infections 257 

Antimicrobial Agents 257 

Rocky Mountain Laboratory 257 

Tularemia 258 

Rabies 258 

Rickettsial Diseases 258 

Transmission of Disease 259 

Tick-Borne Diseases 259 

PLT Group 260 

Progressive Viral Disease 260 

Allergy and Immunology 261 

Tuberculosis 261 

Bordetella Pertussis Antigens 261 

Endotoxins 262 

Microorganisms 262 

Middle Atlantic Research Unit 262 

Hemorrhagic Fever 262 

Arbovirus 262 

Histoplasmosis 263 

NATIONAL INSTITUTE OF MENTAL HEALTH 265 

Introduction 265 

Clinical Investigations 266 

Addiction Research Center 268 

New Analgesic Addiction 270 

Acute and Chronic Intoxication 271 

Alcohol, Barbiturates, and Related Drugs 271 

Biochemistry of Addiction 272 

Neurophysiology and Neuropharmacology of Intoxication 273 

Psychology of Addiction 273 

CNS Depressants 275 

Opiate Addiction 275 

Human Experimentation 277 

Social Science 278 

Chemical Pharmacology 279 

Laboratory of Socio-Environmental Studies 279 

Family and Personality Development 280 

Clinical Settings and Their Patients 282 

Social Psychology of Occupations 283 

Social Science Methodology 284 

Laboratory of Neurophysiology 285 

Section on Limbic Integration and Behavior 285 

Section on Membrane Physiology 286 

Laboratory of Psychology 286 

Section on Neuropsychology 286 

Cortical Mechanisms in Sensation and Perception 287 

Vision 287 

Somesthesis 287 




CONTENTS XVll 

Page 

Cortical Mechanisms in Problem Solving 288 

Sub-Cortical Relations 289 

Section on Aging 290 

Psychophysiology 290 

Higher Cognitive Processes 291 

Section on Perception and Learning 291 

Environmental and Genetic Variables 291 

Perception 292 

Section on Early Development 293 

Children's Perceptions of Parents 293 

Culturally Deprivation 293 

Learning in the Neonatal Dog 294 

Learning and Environment 294 

Section on Personality 295 

Creative Functioning 295 

Conceptual Mediation 297 

Psychedelic Therapy (LSD) 297 

Office of the Chief 297 

Schizophrenia 298 

Psychotherapy 299 

Human Communication 299 

Child Research Branch 300 

Clinical Investigations 300 

Adult Psychiatry Branch 302 

Family Studies 302 

Family Therapy 303 

Intrafamilial Communication 304 

Individual Schizophrenic Functioning 306 

Developmental Aspects 307 

Cross-Culture 307 

Section on Personality Development 308 

Section on Twin and Sibling Studies 310 

Section on Psychosomatic Medicine 313 

Section on Psychophysiology of Sleep 318 

Laboratory of General and Comparative Biochemistry 319 

Section on Proteins 319 

Section on Cellular Regulatory Mechanisms 320 

Section on Alkaloid Biosynthesis 321 

Laboratory of Neurochemistry 321 

Laboratory of Clinical Science 323 

Section on Cerebral Metabolism 325 

Section on Physiology 326 

Sleep 326 

Movement 327 

Section on Pharmacology 328 

Section on Medicine 329 

Section on Biochemistry 331 

Section on Psychosomatic Medicine 332 

Section on Psychiatry 332 

Clinical Neuropharmacology Research Center 335 



xviii CONTENTS 

Page 

Clinical Studies 335 

Clinical Phychiatry 335 

Section on Experimental Psychiatry 336 

Section on Psychosocial Research 336 

Laboratory Studies 337 

Section on Neurophysiology 337 

Section on Neurochemistry 338 

Catecholamines in Urine 338 

Section on Psychopharmacology 340 

Metabolic Studies 340 

Behavioral Studies 341 

Laboratory of Neurobiology 342 

Mental Health Study Center 343 

Office of the Director 343 

Mental Health Planning 344 

Advisory Board 344 

Clinical Study Section 345 

Administration 345 

Service-Oriented Activities 346 

Clinical Studies 348 

Training 350 

Community Projects Section 352 

Community Social Organization 353 

Individual and Group Behavior 355 

Other Activities 359 

Adolescent Process Section 360 

NATIONAL INSTITUTE OF NEUROLOGICAL DISEASES 

AND BLINDNESS 367 

Introduction 367 

Medical Neurology Branch 369 

Clinical Investigation Program 869 

Myopathies 369 

Episodic Weakness 370 

Myasthenia Gravis 370 

Amyotrophic Lateral Sclerosis 370 

Neuroradiology Section 370 

Radiographic Diagnosis : 370 

Radiation Dosimetry 371 

Isotopic Diagnosis 371 

Neuropharmacology Section 871 

Surgical Neurology Branch 372 

Cerebral Trauma 372 

Developmental Defects 372 

Epilepsy 873 

Involuntary Movements 374 

Cerebral Edema 374 

Brain Tumor 375 

Language and Memory 375 

Low Temperatures 376 



CONTENTS Xix 

Page 

Microbial Analysis 476 

Summary 376 

Ophthalmology Branch 377 

Physiology of Vision 378 

Retinal Disease 379 

Uvea 380 

Intraocular Pressure 380 

Cornea 382 

Lens 382 

Ocular Changes in Systemic Disorders 383 

Electroencephalography and Clinical Neurophysiology Branch 884 

Clinical Diagnostic Service 384 

Research Activity 384 

Future Programs 386 

Laboratory of Neuroanatomical Sciences 387 

Sensory Systems 387 

Auditory System 387 

Olfactory System 388 

Visual System 389 

Neuromuscular Relationships 390 

Central Nervous System 391 

Chemical Transfer Across Brain 392 

Degeneration and Regeneration 393 

Future Program 394 

Laboratory of Neuropathology 395 

Laboratory of Neurophysiology 395 

Laboratory of Biophysics 396 

Laboratory of Neurochemistry 398 

Laboratory of Molecular Biology 401 

Nucleic Acids 401 

Effects of Hydroxylamine 401 

Structure of Chromosomes 401 

Virus-Induced Mutants 402 

Mutation in Evolution 402 

Control Mechanisms and Differentiation 402 

Grermination 402 

Synchronized Bacteria 403 

Bacterial Cell Walls 403 

Goals 404 

Laboratory of Perinatal Physiology 404 

Physiological Psychology 405 

Comparative Neurology 406 

Developmental Neurology 406 

Experimental Neuropathology 406 

NATIONAL INSTITUTE OF DENTAL RESEARCH 411 

Introduction 411 

Laboratory of Microbiology 411 

Oral and Pharyngeal Development Section 411 

Human Genetics Branch 412 



XX CONTENTS 

Page 

Epidemiology and Biometry Branch 413 

Dental Caries 414 

Periodontal Disease and Skeletal Disturbance 417 

Calcification, Growth, and Development 419 

Proteins and Enzymes 423 

Oral Lesions: Diagnosis and Treatment 425 

DIVISION OF BIOLOGICS STANDARDS 429 

Introduction 429 

Laboratory of Bacterial Products 430 

Standards 431 

Research 431 

Cholera Vaccine and Vibrios 431 

Pertussis Vaccine 431 

Tetanus Toxoids 432 

Pleuropneumonia-like Organisms 432 

Tuberculin 432 

Poison Ivy 432 

Laboratory of Biophysics and Biochemistry 432 

Research 432 

Microbiol Biophysics 432 

Analytical Chemistry 433 

Electron Microscopy 433 

Control Activities 433 

Laboratory of Blood and Blood Products 433 

Intramural Research 433 

Control Activities 433 

Extramural Research and Training 434 

Laboratory of Control Activities 434 

Control Activities 434 

Research 436 

Laboratory of Pathology 436 

Laboratory of Viral Immunology 437 

Rubella 437 

Measles 437 

Poliomyelitis 437 

Smallpox 438 

Mumps 438 

Virus-Induced Neoplasms 438 

Cell Cultures 438 

Laboratory of Virology and Rickettsiology 439 

Respirovirus Studies 439 

Antimicrobial Agents 439 

Viral Hepatitis 439 

Rickettsial 439 

Arboviruses 439 

Avian Leukosis Virus 440 

Safety Testing 440 

Appendix 440 

Control Testing 440 




NATIONAL CANCER INSTITUTE 



IINTRODUCTION 



The Clinical Director has prepared a brief 
summary of the specific activities in the clini- 
cal branches which he wishes to call attention 
to from the clinical' research programs of the 
National Cancer Institute. The reports of the 
laboratory chiefs should be consulted for spe- 
cific details. At this time attention should be 
called to the broad study of acute leukemia. 
This program has been given emphasis by a 
special appropriation of the Congress in the 
amount of $10 million. This has permitted an 
expansion of the efforts to identify a specific 
virus in the etiology of human acute leukemia, 
to develop additional research support for the 
study of acute leukemia in man and in experi- 
mental animals and to provide a means for ad- 
ditional research in the clinical management of 
patients with acute leukemia. 

Studies of acute leukemia range from studies 
in the chemistry of the virus, through the nu- 
merous leukemia virus models in the experi- 
mental animal, through the chemotherapy of 
experimental animal leukemia to problems in 
the management and therapy of patients with 
acute leukemia. 

A specific accomplishment during the last 
year is the remarkable increase in remission 
rate of acute leukemia in adults. The remission 
rate now reaches the order of 60 percent as 
contrasted to 5-10 percent 1-2 years ago; this 
by virtue of combination chemotherapy. The 
beginnings of studies on reverse isolation also 
hold considerable promise in the study of the 
clinical management and particularly in the 
ability to eliminate infection as a cause of com- 
plicating factors. 

One of the very interesting aspects of the 
special appropriation for acute leukemia has 
been the creation of a large coordinated effort 
with approximately eight program chairmen, 
each of whom has a committee for the pro- 
gramming of research in a specific area. 



While this specific effort in acute leukemia 
can be easily identified, the National Cancer 
Institute intramural research programs do en- 
compass a broad range of activities in basic 
and clinical research in cancer. These range 
from studies of the genetic factors effecting the 
incidence of tumors in the mouse through stud- 
ies of tissue culture, and the chemistry of vi- 
ruses to clinical studies of the chemotherapy of 
trophoblastic disease and the use of radical 
surgery for tumors of the head and neck and 
uterine cervix, and to studies of the biochem- 
istry and physiology of man as altered by a 
malignant tumor. Again attention is called to 
the reports of the laboratory and branch chiefs 
for details of the specific research accomplish- 
ments. 

LABORATORY OF BIOCHEMISTRY 

Cytochemistry Section 

Dr. D. Burk, Dr. M. Woods, and Mr. J. 
Hunter believe that the insulin: anti-insulin 
regulation of glucose phosphorylation in the 
hexokinase reaction plays a major role in con- 
trol of metabolism in normal and malignant 
cells. Previous work had demonstrated that the 
presence of this mechanism in melanomas, 
mammary cancer, Ehrlich ascites tumor (weak 
control), human myeloid and lymphocytic 
leukemias, plasma cell tumor of mice, rabbit 
neutrophils and total bone marrow prepara- 
tions, mouse peritoneal macrophages and 
spleen, tissue culture fibroblast cell lines and 
sarcomas derived therefrom, and mouse kidney 
and mouse brain but not liver. With liver, as in 
the case of brain, there have been numerous 
reports denying the direct action of insulin in 
vitro. The glycolytic actions of normal mouse 
and rat liver of a remarkable series of chemi- 
cally-induced rat hepatomas (developed by Dr. 
Harold P. Morris) have been studied. Previous 
findings as to the importance of the DPN/ 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



DPNH2 ratio in the triosphosphate dehydroge- 
nase reaction as a secondary rate-limiting step 
in glycolysis have been found to be applicable to 
liver. In all cases, detailed analyses showed 
that the glycolytic activity of the hepatomas 
exceeded that of the corresponding host liver, 
and also exceeded the average rate of livers 
from a series of tumor-free rats. Even the most 
slowly-growing and least anaplastic of the 
hepatomas glucolyzed more than the corre- 
sponding host livers. It was possible to show 
that in the liver-hepatoma spectra there is a di- 
rect correlation between the rate of glucolysis 
and tumor growth rate, and that these are re- 
lated to the susceptibility of glucose utilization 
to anti-insulin regulation. As would be expect- 
ed, the affinity for glucose was much higher 
(Km much lower) in the tumors than in liver, 
and was proportional to the degree of anti-in- 
sulin restraint. In both liver and the hepa- 
tomas, in vitro stimulation of glucolysis by 
glucagon-free insulin was obtained at critical 
glucose concentrations. Further studies of endo- 
toxin action on carbohydrate balance (blood, 
sugar, liver glycogen), in vivo and in vitro, 
continue to support previous conclusions that 
these toxins exert an insulin-like action on 
mammalian tissues. 

Examination of the effect of anti-cancer 
agents and hormonal systems that inhibit 
cancer metabolism and growth have been cen- 
tered on those compounds which influence the 
DPN/DPNH2 ratio at the triosephosphate de- 
hydrogenase (TPDH) locus and were extended 
to various tissues, including liver and the 
Morris series of rat hepatomas. Under in vitro 
conditions commonly employed in determining 
the effects of chemotherapeutic agents on cells, 
the TPDH locus in certain tissues, particularly 
liver and hepatomas, may become a rate limit- 
ing reaction masking the true glycolytic poten- 
tial of the cells. The interaction between liver 
or hepatoma cells and acridine dyes plus visible 
light was investigated. As in Ehrlich ascites 
tumor cells, DPNHa-induced inhibition of he- 
patic glycolysis could be reversed by excess 
(10-* M)acriflavine in visible light with oxida- 
tion of DPNH2. These new results obtained with 
liver and hepatomas should be helpful in under- 
standing and maximizing the action of chem- 
otherapeutic agents that involve loci of coen- 



zyme I (DPN) function, and in more accurately 
evaluating the effects of agents which act on 
glycolysis. 

Studies on the effects of light-sensitized dyes 
on the metabolism and growth of tumors indi- 
cate that white light and methylene blue, act- 
ing together, greatly increase the DPN re- 
quirement for proper metabolic function of 
ascites cancer cells. The increased require- 
ment of DPN by cells in the presence of methy- 
lene blue and light can be satisfied in varying 
degrees by exogenous DPN, depending upon dye 
concentrations below 10"* M. The increased re- 
quirement for DPN may be due to peroxide f or- 
maton with concomitant coenzyme destruction, 
to increased competition between DPN and 
photosensitizing dye for available hydrogen, or 
both. Glycolysis seems to be more sensitive 
than respiration to the photodynamic effects of 
these dyes, and in this way, the photodynamic 
action is similar to the action of hydrogen per- 
oxide and X-irradiation. Under proper condi- 
tions, photodynamic action of photosensitive 
dyes, like X-irradiation, may prove useful in 
cancer chemotherapy. 

Biochemical studies on normal and leukemic 
leukocytes are being directed by Dr. W. H. 
Evans to determine the biochemical factors 
regulating the growth, maturation and phago- 
cytic capacity of leukocytes, especially with 
regard to the arrested maturation and de- 
creased phagocytic activity of leukocytes from 
myelogenous leukemia. It has become necessary 
to develop techniques for the rapid fractiona- 
tion of cell types from bone marrow and blood 
so that the biochemical investigation of cell 
maturation can proceed. One of the major ob- 
stacles to successful cell fractionation has been 
overcome. This has been the tendency of dis- 
persed marrow cells to reaggregate. Treat- 
ment of bone marrow cells with DNAase and 
trypsin followed by washing and resuspension 
of cells in a medium containing sodium acetate 
suppresses aggregate formation. A new sedi- 
mentation method is now being developed in 
collaboration with Dr. E. A. Peterson to re- 
solve and separate these enzyme-treated bone 
marrow cells. Studies on the relative roles of 
catalase and myeloperoxidase in leukocyte per- 
oxidative metabolism suggest that catalase 
serves as a peroxidative enzyme in intact cells 



NATIONAL CANCER INSTITUTE 



but only accounts for part of the total peroxi- 
dative metabolism. The extent to which myelo- 
peroxidase contributes to the total peroxidative 
metabolism of intact leukocytes is not yet clear. 

Nucleic Acids Section 

In attempting to explain the accumulation of 
dCDP-choline and dCDP-ethanolamine in the 
Novikoif hepatoma, studies of the deoxyribose- 
containing compounds in normal, regenerating, 
and cancer tissues have been advanced on sev- 
eral fronts by Dr. W. Schneider and Dr. W. G. 
Fiscus (Research Associate). The preparation 
of labeled nucleotides has presented difficulties 
when chemical methods were used. However, 
this problem has now been solved by the use of 
enzymes present in rat liver supernatants. In 
the presence of ATP and phosphoglycerate, 
yields of 55 percent or more of H^ or C"-la- 
beled CDP-choline, dCDP-choline, CDP-etha- 
nolamine, and dCDP-ethanolamine were ob- 
tained in a two hour incubation with rat liver 
supernatant fractions. With rat liver superna- 
tants as source of the phosphorylase, it was also 
possible to synthesize phosphocholine C" and 
phosphoethanolamine-C" from ATP-choline 
and ethanolamine in yields approaching 100 
percent. 

The two enzyme fractions that are involved 
in the synthesis of dCDP-ethanolamine and 
dCDP-choline can be differentiated by their 
isoelectric points, by their precipitability by 
salts, and by the fact that the former enzyme 
shows no activation phenomena. The latter en- 
zyme, phosophocholine-cytidyl transferase, has 
been shown to be localized in the supernatant 
of rat liver and hepatoma homogenates and 
shows a remarkable increase in activity upon 
aging at 0° or 37°. The studies of this activa- 
tion phenomena have indicated that the activa- 
tion is induced by the phospholipid fraction of 
rat liver. A commercial preparation of egg lec- 
ithin which was obviously degraded was also 
highly active, but highly purified egg lecithin 
which had been protected from degradation 
showed little activating until it had been ex- 
posed to unfavorable conditions and even then 
was greatly inferior to the most active egg lec- 
ithin or rat liver phospholipid preparations. 
Studies of DNA synthesis in hepatoma have 



suggested that the mechanism of incorporation 
of dCDP-ethanolamine into DNA was through 
dCTP rather than by direct incorporation. 

The discovery that altered phospholipids ac- 
tivate the CDP-choline and dCDP-choline 
synthesizing enzymes is of fundamental inter- 
est and may be a most important finding. Al- 
though it is not yet clear what alteration is nec- 
essary in the phospholipid before it can serve as 
an activator it suggests that an unusual mech- 
anism is operative in providing a constant 
supply of lecithin in the cell. Thus, it is visual- 
ized that as lecithin is broken down in the cell, 
the breakdown product or products activate the 
enzyme responsible for synthesis of the imme- 
diate precursor of lecithin, CDP- or dCDP-cho- 
line. This would provide a unique function for 
phospholipids and another metiiod for main- 
taining cellular lecithin levels. 

Several recent reports have claimed that 
DNA is present in Neurospora, rat liver and 
kidney, and calf heart mitochondria in 
amounts less than one percent of the total tis- 
sue DNA. In order to verify this finding and 
establish that contamination was not present, 
mitochondria were isolated from rat liver. The 
DNA fraction of the liver was isolated, freed 
of RNA by treatment with alkali, and hydro- 
lyzed to the component bases. The nucleotide 
bases were separated by paper chromatog- 
raphy. This mitochondrial DNA showed a 

A -F T 

small but significant difference in the 

G + C 

ratio as compared to nuclear DNA. Confirma- 
tion of the presence of DNA in mitochondria 
add to the growing body of evidence that these 
organelles contain their own DNA and thus 
may be self-duplicating and transmitters of 
hereditary information in their own right. 
Since tumors are known to be deficient in mito- 
chondria, it suggests that the point of attack of 
a carcinogen or carcinogenic virus may be at 
the level of mitochondria. 

The metabolism of the acid soluble deoxyri- 
bosyl compounds of animal tissue has been stud- 
ied by Dr. J. Rotherham who has found that 
the total amount of deoxyribosyl compounds 
excreted in the urine of rats was lower when 
the animal was fed a synthetic diet containing 
no preformed source of these compounds than 
when animals were fed laboratory chow. At 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



least a part of these endogenously-f ormed com- 
pounds were probably not derived from in vivo 
degradation of deoxyribonucleic acid. The con- 
centration of deoxyribosides in the interstitial 
fluid of two different tumors was lower than in 
the circulating fluid and the concentrations in 
the interstitial fluid in a third type of tumor 
and in normal subcutaneous tissue were the 
same as in normal blood. Studies have also 
been initiated comparing the structural proper- 
ties and metabolism of the nucleic acids and 
nucleoproteins of cultured cells and of tumors 
derived therefrom. 

Dr. R. Kielley's research on the cellular mech- 
anism and the control of DNA synthesis has 
been concerned with the factors influencing the 
synthesis of thymidine triphosphate (TTP) in 
normal and tumor cells, i.e., the enzymatic con- 
version of thymidylate (TMP) to high-energy 
phosphorylated forms by the action of TMP- 
kinase. The particle-bound TMP-kinase pre- 
viously found in the twice-washed mitochon- 
drial fraction of mouse liver, has now been 
more precisely localized in the "large granule" 
fraction which corresponds more nearly to in- 
tact mitochondria free of lysosomal and micro- 
somal contaminants, thus supplying confirma- 
tive evidence that intact mitochondria contain 
bound TMP-kinase and tends to discount the 
possibility of non-specific adsorption or enzyme 
concentration in some less well-defined particle 
in the crude mitochondrial fraction. In mouse 
liver, the enzyme causing degradation of TMP, 
the TMP-nucleotidase, was found in greatest 
amounts in the soluble fraction and not in the 
microsomal fraction. The ability to recover 
TMP-kinase from mitochondria of mouse liver 
probably is due to the weak mitochondrial nu- 
cleotidase activity. It has been shov^m that the 
TMP-kinase reaction in hepatoma extracts can 
be more rapidly limited by substrate depletion 
by addition of the soluble fraction of mouse 
liver which contains most of the TMP-phospha- 
tase activity. Addition of other liver cell frac- 
tions caused little effect. The rate of a kinase 
reaction remained linear provided there re- 
mained excess TMP and provided ATP re- 
synthesis was maintained with a high degree 
of efficiency. When ATP resynthesis was sup- 
ported by glycolysis, complete recoveries of the 
combined kinase activities of mouse liver and 



hepatoma could be obtained. On the other hand, 
when conditions of ATP resynthesis by oxida- 
tive phosphorylation were permitted, only 50 
percent of the combined kinase activities could 
be obtained. These plus more complete kinetic 
data suggest that in addition to ATP concen- 
tration itself, which was fairly well maintained 
by oxidative phosphorylation even when kinase 
activity was markedly inhibited, the break- 
down and dismutation products, ADP and 
AMP might act as inhibitors of kinase reac- 
tion. These results provide some evidence that 
the state of equilibrium of the adenylate sys- 
tem, ADP*=?AMP + ATP, may be an important 
factor in the regulation of DNA precursor 
synthesis in normal and tumor cells. 

As part of a continuing study on the func- 
tion and relationships of the structure and se- 
quences of nucleic acids, work on the nucleotide 
distribution in nucleic acids, on the develop- 
ment of fractionation procedures, and on the 
preparation and characterization of nucleotide 
sequences has been continued by Drs. G. W. 
Rushizky and H. A. Sober. In collaboration 
with Dr. B. P. Doctor of the Walter Reed 
Army Medical Center, nucleotide and oligonu- 
cleotide composition and analysis of two well- 
separated serine transfer RNAs was accom- 
plished. Fractionation of pancreatic RNase 
digests showed that the two components (serine 
II and serine III RNA) had identical base ra- 
tios and mono- to tetranucleotide composition. 
However, sej-ine II RNA had twice as much 
ApUp than did serine III RNA. Conversely, 
the serine III RNA contained twice as much 
(AG) GU than did serine II RNA. The latter 
tetranucleotide was identified as being either 
AGGU or GAGU. While the nucleotide se- 
quence analyses included only about 70 percent 
of the total RNA of each component, repetition 
of hydrolysis with another RNase may provide 
the remaining 30 percent of RNA components. 

Continued improvement in fractionation 
procedures of larger oligonucleotides has been 
accomplished by the use of buffers containing 7 
M urea at pH 2.7 and at pH 4. At the lower pH, 
fractionation occurs according to the net nega- 
tive charge of the oligonucleotides, i.e., in 
RNase Ti digests which terminate in G, on the 
A+C/U ratio. At pH 4, taking advantage of 
the difference in ionization of adenylic and cyt- 



NATIONAL CANCER INSTITUTE 



idylic acids; separations based on the number 
of A and C residues in oligonucleotides of like 
chain length can be accomplished. In this 
manner, tetra through decanucleotides have 
been examined in RNA Ti digests of MS 2 
ENA. 

Oligonucleotides isolated and purified in this 
manner have been used as primary standards 
for the computer library in studies of computa- 
tional spectral analyses that are being 
performed in collaboration with Dr. A. W. 
Pratt and Mrs. J. N. Toal (Laboratory of Phys- 
iology, NCI) . The recognition, purification and 
characterization of nucleases with hydrolytic 
activity directed towards specific nucleotide 
linkages has turned to the isolation of a group 
of DNases that are free of RNase activity and 
non-specific phosphodiesterase activity. These 
enzymes have strict requirements for metals 
and hydrolyzed DNA as endonucleases. Kinetic 
experiments have verified that, in common 
with other known DNases, these new DNases 
can only hydrolyze one DNA chain at a given 
site in the double strand of helix. The specific- 
ity of the DNases isolated resembles that of 
pancreatic DNase since only mono- and dinu- 
cleotides remain at the end of digestion. A mix- 
ture of enzymes with closely-related metal re- 
quirements and pH optima results in 3' phos- 
phates of mononucleosides and facilitates the 
determination of base ratios in DNA. No 
DNases with unique specificities have yet been 
found. Several RNases that were previously 
isolated and characterized as metal-requiring 
enzymes have been employed for the hydrolysis 
of UNA since addition of EDTA provides a 
very mild procedure for the reversible termina- 
tion of enzymatic hydrolysis under conditions 
where phenol extraction would interfere. The 
use of these non-specific RNases also provides 
means for the preparation of 2',3'-cyclic ter- 
minal-ended oligonucleotides. 

A simplified procedure has been devised for 
the large scale preparation of E. coli phage MS 
2. From 300 liters of E. coli, up to 19 g of 
phage can be obtained. Previously used proce- 
dures involve a series of steps which gave 
lower yields and which were difficult to scale 
up. 

In collaboration with Dr. C. W. Lees (Re- 
search Associate) methods are being sought 



for the purification of the Moloney Virus (Dr. 
J. B. Moloney, Laboratory of Viral Oncology, 
NCI). It has been found that viral activity 
emerged in the excluded fraction of G-200 
Sephadex columns and was adsorbed on DEAE- 
cellulose. The activity could be eluted by in- 
troducing sodium chloride in concentrations 
ranging from 0.03 M to 0.06 M. Preliminary 
experiments with aqueous polymer, liquid-liquid 
partition indicate that viral activity is concen- 
trated in the lower phase of a 5.35 percent 
sodium dextrose sulfate — 8.0 percent poly- 
ethylene glycol system. 

Oligoribonucleotides from RNA and syn- 
thetic ribonucleotide polymers have been used 
to study the formation of polylysine-RNA com- 
plexes (in collaboration with Dr. A. Yaron, Vis- 
iting Scientist, and Dr. S. Schlossman, Re- 
search Associate). Digestion of the soluble 
complex results in the formation of a precipi- 
tate. This insoluble RNA-polylysine complex 
consists of a fragment of the original RNA 
chain and polylysine in a lysine: nucleotide ra- 
tio of 1. The complex can be dissociated and the 
protected RNA sequence recovered and ana- 
lyzed. No base specificity has yet been found 
but the protected sequence bears a direct rela- 
tionship in chain length to the chain length of 
the polylysine used for protection. Thus, treat- 
ment with polylysine, ii=100, results in a pro- 
tected RNA sequence of h=99; the use of oli- 
golysine, n=13 resulted in RNA chain length 
of n==16. 

Deoxyribonucleotides from partial and com- 
plete digests of calf thymus DNA have been 
used for specificity studies with new DNases. 
Other deoxynucleotides prepared from rat 
spleen have been used to check their function 
as stimulants of plasma cell formation. Prelim- 
inary results obtained with Dr. M. Feldman, 
(the Weizmann Institute of Science, Rehovoth, 
Israel) indicate that deoxyribonucleotides 
(ii^lO) were able to reactivate the immune re- 
sponse in thymectomized. X-ray irradiated 
adult mice. 

Nutrition and Carcinogenesis Section 

Intensive examination of the enzymatic and 
metabolic characteristics of "minimal deviation 
hepatomas" and tumors of different growth 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



rate has been continued by Dr. H. P. Morris 
with the collaboration of some 25 laboratories 
throughout this country and abroad. The gen- 
eralizations reported last year still remain true 
"that no two minimal deviation tumors are 
alike, and that their enzyme, metabolic and 
physiologic patterns show striking individual 
differences. Furthermore, no single biological 
or biochemical abnormality has been uncovered 
which is common to all of the "minimal devia- 
tion hepatomas." The extensive examination of 
these tumors can be summarized as indicating 
that the minimal deviation hepatomas form a 
spectrum of neoplasms, each differing from the 
other in enzyme level and metabolic pattern. 

The original minimal deviation tumor 5123 
can no longer be considered a unique trans- 
plantable liver tumor. New groups of such 
transplantable hepatomas including several 
new highly-differentiated hepatomas have been 
developed and are now being studied biochemi- 
cally and histologically. Variations in the chem- 
ical structures of the inducing carcinogens 
have produced tumors at different sites and 
with variable rates of growth. Three rapidly- 
growing solid transplantable hepatomas have 
been converted by Dr. S. Odashima (Visiting 
Scientist, Laboratory of Pathology, NCI) to 
the ascites form but similar conversions have 
not succeeded with 17 others. None of the 
slowly-growing tumors have been converted to 
the ascites form. 

Hepatomas in rats maintained on high cho- 
lesterol diets continue to synthesize cholesterol, 
implying the loss of the normal feedback con- 
trol mechanisms on cholesterol synthesis, but 
to a variable extent; the slowest-growing tu- 
mor synthesized the most cholesterol. Slow- 
growing hepatomas have zero or borderline lev- 
els of glucokinase which are not correlated 
with growth rate. The feedback inhibition of 
thymidine kinase was inhibited by TTP and 
dCTP in the slowly-growing hepatomas. The 
activity levels of three glycolytic enzjrmes, glu- 
cokinase, phospho-fructokinase, and pyruvic 
kinase were elevated in rapidly-growing tu- 
mors suggesting that these tumors are geared 
to straight glycolysis whereas some of the en- 
zymatic activities of slowly-growing tumors 
deviate in the opposite direction. Several 
slowly-growing hepatomas readily oxidized 



fatty acids suggesting that fatty acid oxidation 
may be the major source of energy for slowly- 
growing hepatomas that have slight capacity 
for glycolysis. 

Studies on the effect of carcinogens on the 
induction of gastric tumors in rats have shown 
that the combined effect of two carcinogens, 
2,7-FAA and 3-methylcholanthrene were addi- 
tive. Gastric adenomas were induced in rats 
even though the injection of 2-FAA had oc- 
curred at a site remote from the stomach, 
implying that the carcinogen or its metabolite 
must have penetrated the stomach wall from 
the peritoneal cavity or from the blood stream. 
This observation suggests that an experimental 
test system is at hand in which the mechanism 
of gastric carcinoma can be studied. 

Additional studies on transplantable hepa- 
tomas have been performed by Drs. T. T. Otani 
and H. P. Morris, in which the relative distri- 
bution of the two isozymes of glutamic-oxalic 
transaminase has been determined. The effect 
of extrinsic agents on their relative distribu- 
tion has also been studied in attempts to corre- 
late the isozyme patterns and the growth rates 
of several transplantable hepatic tumors. In 
five tumors examined the two isozymes of glu- 
tamic-oxalic transaminase had electrophoretic 
mobilities that were similar to each other and 
to those of the normal liver glutamic-oxalic 
transaminase. Studies of the Km values of the 
anionic component of the two isozymes has 
shown that this component appears to be the 
more variable, one with the values of the an- 
ionic components tending to approach the Km 
of the anionic component of the normal liver 
enzyme as the growth rate of the tumor in- 
creases. 

Examination, by Dr. H. P. Morris, Dr. H. 
Dyer and Mrs. B. P. Wagner of the role of 
tryptophan and indole in the induction of 
bladder tumors in rats ingesting N-2-fluorenyl- 
acetamide (2^FAA) has showTi that bladder 
tumors can be produced in Fischer strain rats 
without the addition of tryptophan. In a strain 
of genetically-jaundiced rats ingesting 0.025% 
2-FAA as many liver tumors developed as in 
non- jaundiced animals of the same genetic 
background. Lower levels of 2-FAA are now 
being fed to determine whether an overwhelm- 
ing exposure to the carcinogen had been used. 




NATIONAL CANCER INSTITUTE 



Biochemical studies in experimental cancer 
by Dr. H. Dyer and Mrs. K. Lueders have re- 
volved around the carcinogenesis and metabo- 
lism of 2-FAA and 2,7-FAA. Studies with 
monkeys have shown that both of these carcin- 
ogens can be recovered quantitatively in the ex- 
creta of monkeys. Chromatographic studies of 
metabolites indicate that 2-FAA may not be 
carcinogenic for the monkey. Fecal excretion 
of great amounts of unmetabolized 2,7-FAA 
following oral rather than parenteral adminis- 
tration suggests that this very insoluble com- 
pound should be administered intraperitoneally 
for testing carcinogenicity. 

With rats fed 2,7-FAA for six months, 
slight differences observed in the mucin con- 
tent of the glandular stomach and gastric juice, 
decreased volumes of gastric juice, and a 
higher pH and lower free and total acid com- 
pared with control rats, may reflect early 
changes related to the occurrence of neoplasia 
of the gastric mucosa of rats fed the same diet 
for 10 to 12 months. 2-F-diAA-induced liver 
nodules in rats incorporated decreasing 
amounts of radioactivity from a single oral 
dose of 2-FAA-9-C^^ with progressive devel- 
opment of nodules from early states of hyper- 
plasia to the frank appearance of hepatomas. 
This observation may reflect the protein bind- 
ing of carcinogens by liver and the lack of 
binding by hepatoma proteins. Greater glucu- 
ronyl transferase activity was found in homo- 
genates and microsomal fractions of trans- 
planted, slowly-growing, well-differentiated 
hepatomas than in normal or host livers, and 
less than in fast-growing hepatomas than in 
liver. Kinetic behavior and stability studies 
have demonstrated the similarity of enzyme ac- 
tivities in liver and hepatomas. 

yS-Hydroxy-a-amino acids and their N-acyl 
derivatives are being prepared by Dr. T. T. 
Otani. The free /3-hydroxy-a-amino acids as 
well as their N-acyl derivatives are pretested 
for growth inhibitory action on microbial sys- 
tems to study amino acid metabolism and to 
detect possible anti-tumor action. Compounds 
exhibiting positive action such as the N-acyl 
derivative of jS-hydroxynorleucine are being 
tested on tumors and intact mammalian sys- 
tems and on isolated mammalian cells in cul- 
ture. The small amount of activities resulting 



have indicated that resolution of racemic mix- 
tures and separation of diastereoisomers of 
these /?-hydroxyamino acids are required. The 
resolved diastereoisomers and their N-acyl de- 
rivatives are being prepared, with the hope that 
an increased activity over that shown by the 
racemic mixture will be obtained. This class of 
compounds offers a new group of tumor-inhib- 
iting agents with a mode of action unlike 
those now in use in that they react within the 
cell to release an unnatural amino acid and the 
chloroacetic acid moiety. 

Protein Chemistry Section 

The development of new principles and the 
improvement of older procedures for the frac- 
tionation and characterization of macromole- 
cules of larger structures of biochemical inter- 
est, e.g., ribosomes and whole cells, is of major 
importance and continues to accrue dividends 
by application to specific research problems. 

Such development has resulted in the novel 
micropath sedimentation technique which is 
being used by Drs. E. A. Peterson and W. 
Evans for the separation of leukocytes and 
myelocytes from other leukocytes in guinea pig 
bone marrow and in serum. The early separa- 
tions obtained have been shown to be due to the 
tendency of the mature granulocytes, myelo- 
blasts, and red cell precursors to form aggre- 
gates, a tendency not shared to the same extent 
by red cells and myelocytes. Since such aggre- 
gation would be a major obstacle to the further 
fractionation of leukocytes by any method, con- 
siderable effort has been expended to minimize 
this tendency. A systematic study of the effec- 
tive agents on cellular aggregation suggested 
that nucleoprotein adhering to the cells might 
be a factor. An effective method of pretreating 
the bone marrow cells with a mixture of tryp- 
sin and DNase was adapted from a procedure 
developed by Madden and Burk of this labora- 
tory in 1960 for the preparation of viable 
single cell suspensions from solid tumors. Ad- 
ditional factors in the improvement of the cell 
separation were the use of gentler means of dis- 
persing the marrow, final filtration of the cells 
through porous polyethylene membranes, the 
addition of sodium acetate to the medium and 
the omission of calcium and magnesium. 



8 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



Nearly all the cells of such a preparation 
passed through a slowly-rotating horizontal col- 
umn of small pore polyurethane foam whereas 
most of the leukocytes of the early prepara- 
tions were strongly retarded by large pore 
foams. 

Although some separation has been obtained, 
sufficient resolution has not yet been accom- 
plished. A variety of material in shredded and 
beaded form have been tested. Of all the mate- 
rials tested, so far, IRC-50 oifered the most 
promise in that few cells were adsorbed or 
trapped. Its effectiveness in fractionating the 
cells by sedimentation will be explored in long 
columns. Adsorption of cells to the materials 
tested v/as not correlated with presence of 
charge or its sign. Further examination of par- 
ticles and ion exchange resins for the micro- 
pore sedimentation technique will be continued. 
Preliminary experiments have shown that it is 
simple to coat a polyurethane foam with a pol- 
ymer containing clusters of positive charges 
that can be readily controlled by reasonable 
changes in pH, thus limiting the changes in 
salt concentration that would be necessary for 
elution. 

In an investigation of cytoplasmic nucleopro- 
teins and their role in protein synthesis Drs. E. 
A. Peterson, E. Kuff and L. Kedes (Research 
Associate) have been isolating cytoplasmic-ri- 
bonucleoprotein particles and their protein and 
nucleic acid moieties by a combination of cen- 
trifugal and chromatographic techniques for 
use in metabolic experiments. A comparison of 
liver ribosomes and ribosomes isolated from 
rabbit reticulocytes subjected to the chromato- 
graphic procedures has shown that the reticu- 
locyte ribosomes emerge as a single peak where- 
as the liver ribosomes yield two peaks in the 
sodium chloride gradient with a prominent fi- 
nal peak eluted by trisodium phosphate. The 
reticulocyte ribosomes showed an unchanged 
chromatographic pattern on rechromatography 
whereas the liver ribosomes shifted in position 
from the first to the second peak. When prepa- 
rations of reticulocyte monosomes and penta- 
somes obtained by zonal ultracentrifugation 
were subjected to the chromatographic proce- 
dures, the monosomes and pentasomes emerged 
at different salt concentrations, and these frac- 
tions retained their original ultracentrifugal 



characteristics. When the reticulocytes were 
incubated with the radioactive-labeled amino 
acids and then lysed and subjected to chroma- 
tography, the profile of the radioactive label 
coincided with that of the ribosomes and al- 
most 100 percent of this radioactivity was sed- 
imentable. Centrifugation in a sucrose density 
gradient revealed a high specific activity in the 
polysomes, indicating that the chromato- 
graphic procedures were mild enough to avoid 
disaggregation of the active polysome fractions. 

A comparison of a reticulocyte ribosome iso- 
lated by centrifugation with those obtained by 
chromatography indicated that they were 
nearly identical in their ability to incorporate 
C" leucine in a complete system of co-factors. 
The chromatographically isolated ribosomes, 
however, were free of endogenous co-factors 
whereas ribosomes prepared by centrifugation 
incorporated 60% as much label without added 
co-factors as with them. In an in vitro system 
containing the required co-factors, rat liver ri- 
bosomes (monosomes obtained by this chromat- 
ographic procedure) were able to incorporate 
radioactive labeled phenylalanine when stimu- 
lated by poly-U. This incorporation increased 
in a nearly linear manner with increase in 
poly-U concentration. At the maximum poly-U 
concentration tested (100 gamma per ml) the 
activity of ribosomes prepared chromatograph- 
ically was nearly identical to that of ribo- 
somes (polysomes) obtained by centrifugation 
through a sucrose layer. The latter ribosomes 
were not stimulated to increased activity by 
the addition of poly-U. 

The fact that ribosomes prepared chromato- 
graphically have a lower intrinsic incorporat- 
ing activity than those prepared by centrifuga- 
tion, yet can be brought to the same high level 
of incorporating activity by the addition of co- 
factors makes such ribosomes particularly use- 
ful in the study of the co-factors and the mech- 
anisms involved in amino acid incorporation. 

It has been observed that nucleic acids that 
can be presumed to be single stranded, such as 
denatured DNA and the most rapidly-labeled 
RNA, are adsorbed to ECTHAM-cellulose very 
strongly and emerge in the final NaaPOi eluate. 
Preliminary experiments indicate this fraction 
can be released from the column by increased 



NATIONAL CANCER INSTITUTE 



temperature, promising a very useful means 
for their isolation. 

As part of a continuing study on the homo- 
geneity and structure of proteins Dr. N. Cum- 
mings (Research Associate) and Dr. H. A. 
Sober have begun to study the phenomenon of 
cryo-precipitation. Cryoglobulins are a class of 
serum proteins found in various diseases, most 
notably multiple myeloma, which are charac- 
terized by precipitation in the cold, cryo-precip- 
itation. This has important clinical aspects, 
producing symptomology and loss of biologic 
function often vital to the patient. The mechan- 
ism of precipitation of proteins as a function 
of temperature is important and fundamental. 

Further studies of the metalloproteins of hu- 
man serum, undertaken in collaboration with 
Dr. B. Vallee (Harvard University) have been 
concerned with the nature and possible role of 
magnesium as a protein-bound metal. Fraction- 
ation of human serum on DEAE-cellulose has 
indicated that magnesium is associated with 
proteins of gamma-globulin mobility. However, 
dialysis, electrophoresis, Sephadex and polya- 
crylamide gel fractionation point to a loose 
binding between the metal and the protein, i.e., 
the existence of a magnesium-protein complex 
rather than a metalloprotein per se. Recently a 
technique combining analytical and prepara- 
tive ultracentrifugation developed in collabora- 
tion with Dr. E. Kuff has been used in an effort 
to study the distribution of magnesium and 
serum without introducing electrical or ionic 
forces that may have previously disturbed or 
displaced the original metal-protein equilib- 
rium. Preliminary results indicate that about 
70% of serum magnesium is unbound and that 
the remaining 30% is bound to the albumin 
fraction. The magnesium-albumin association 
is relatively weak since the magnesium can be 
easily displaced to other serum protein frac- 
tions. 

The properties of poly-a-amino acids, model 
substances closely analogous to proteins, are 
being studied by Dr. H. A. Sober and Dr. A. 
Yaron (Visiting Scientist) in collaboration 
with Drs. A. Berger and E. Katchalski of the 
Weizmann Institute of Science, Rehovoth, Is- 
rael. Separation, isolation, and characteriza- 
tion of lysyl oligopeptides, which are of inter- 
est as histone analogs, was accomplished, and 



one quarter to one gram quantities of the oli- 
gomers from the dimer to the decamer were ob- 
tained. The series has been extended to other 
members of the series in yields of approxi- 
mately 30 mg each. These individual oligomers 
of lysine of known chain length and purity were 
used for the preparation of a,N-DNP-and car- 
bobenzoxy-derivatives for rotatory dispersion 
and spectral studies and to study the effect of 
chain length on development of color with nin- 
hydrin. Potentiometric titrations on this series 
of compounds have shown the gradual decrease 
with increasing chain length of the apparent 
pK of the £-amino group from 10.6 to 9.9. Opti- 
cal rotatory dispersion studies with Dr. G. Fas- 
man of Brandeis University have shown that 
at pH 12 conformation (helicity) begins to ap- 
pear with the octamer and increases with in- 
creasing chain length. Organic solvents stabi- 
lize the helix form with oligomers of chain 
length 15 or more but have no effect on long 
chain length polylysines (n=100) and appear 
to destabilize the "helix" forms of the octa-, 
nona- and decalysines. The higher polylysines 
(h=100) aggregate reversibly and form precip- 
itates at higher temperatures (40°), the rate 
of aggregation increasing with temperature. 
The lower members of the series on the other 
hand did not aggregate or associate. The differ- 
ent effects of organic solvents and temperature 
on the lower and higher members of the series 
suggests that while hydrogen bonds are prob- 
ably the dominant stabilizing factor in the 
long chain molecules, hydrophobic side-chain 
interaction is a major factor in the short heli- 
cal structures. 

Interaction of polylysines with polynucleo- 
tides and nucleic acids has led to the formation 
of nuclease-resistant insoluble complexes from 
which oligonucleotides sigments can be ob- 
tained. The length of the protected RNA seg- 
ment is directly related to the length of the oli- 
golysine allowed to interact. Oligolysines of 
known chain length have been used in inhibi- 
tion studies of immunological phenomena to de- 
termine the size of the antibody site. In collab- 
oration with Dr. M. Sela and Dr. R. Arnon of 
the Weizmann Institute, it has been shown that 
maximum inhibition is obtained with oligoly- 
sines of the same chain length as that of the 
immunizing antigen. 



10 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Mono-substituted hapten oligolysines (a,N- 
DNP-oligo-L-lysines) prepared and used in im- 
munological studies have provided a chemi- 
cally-defined homogeneous series Avhich ranges 
from non-antigenicity to full antigenicity. 
These purified, chemically-defined antigens, dif- 
fering in size by increments of one lysyl resi- 
due have been used by Dr. S. Schlossman (Re- 
search Associate) in studies of the chemical 
basis of antigenicity and the basis of the heter- 
ogeneity of antibody. In Hartley Strain and 
Strain 2 guinea pigs it has been found that the 
octamer and larger hapten oligopeptides are 
antigenic; smaller peptides are not antigenic. 
The L-configuration of the a:,N-DNP-haptens 
were essential for antigenicity. Both delayed 
and immediate sensitivity reactions, which 
were easily separated in these two strains of 
guinea pigs, had the same structural require- 
ments and were both obtained from the same 
antigen. 

The extracellular RNase from B. subtilis is 
being used by Dr. R. W. Hartley, Jr. and Dr. C. 
W. Lees (Research Associate) in their study of 
the relation between the structure of an en- 
zyme, its function, and its synthesis. The small 
size, stability, absence of disulfide bridges, and 
easily measured activity of this enzyme, plus 
the fact that the genetic apparatus of this bac- 
terium has been widely studied and manipulat- 
ed, have suggested this ribonuclease as an ex- 
cellent choice for such a study. 

Nearly half of a gram of purified enzyme has 
been produced. The current product gives a 
single peak in gel filtration chromatography 
and only very faint bands of impurities can be 
seen by disc electrophoresis at pH 4.3 and at 
pH 2. Precipitating antibodies to the enzymes 
have been obtained and have shown that two 
bands appear in Ouchterlony double diffusion. 
Both antigens are found in the enzyme band in 
the pH 4.3 disc electrophoresis run. However, 
preliminary evidence suggests that only one of 
the bands possesses RNase activity. 

Study of the heat and pH stability character- 
istics of the enzyme has temporarily foundered 
due to the relatively large amounts which can 
be irreversibly adsorbed to glass and plastic 
surfaces. The rate and perhaps the amount of 
such losses depend on the salt concentration, 
pH, and temperature and on the specific cations 



present. All bivalent cations greatly reduce the 
rate of loss. Iron, however, affords protection 
which exceeds all others by several orders of 
magnitude. In the presence of 10"* M FeClj no 
detectable loss on glass surfaces can be found 
at room temperature after 48 hours. 

RNase from an independently isolated B. 
subtilis strain has been partially purified and 
shown to resemble that of the original strain 
very closely, including immunochemical cross 
reaction. A number of mutants have been ob- 
tained that are deficient in the production of 
extracellular amylase and protease. One double 
mutant, producing neither of these enzymes, 
produces normal amounts of the ribonuclease. 
A report from another laboratory that a pow- 
erful inhibitor of the enzyme may be obtained 
from the cells of the same B. subtilis strains 
that produce the enzyme has been confirmed 
and efforts to isolate the inhibitor are under 
way. 

The cytochemical organization of normal 
and malignant cells is being examined by Drs. 
E. Kuff and W. Hymer and Mr. N. Roberts, 
with special reference to the problems of the 
duplication of intracellular components. Trans- 
plantable plasma cell tumors in mice are pres- 
ently the main objects of study since they pro- 
vide a system for investigating relationships 
between a differentiated function (secretory 
protein synthesis) and neoplastic cell growth. 
Attention has been focused on the isolation and j 
characterization of cytoplasmic RNA from the ^ 
RPC-20 solid tumors. 

The selective loss of a quantitatively small 
but metabolically active type of RNA when 
conventional phenol extraction procedures 
were used, led to the development of a novel 
method which permits direct sucrose gradient 
analysis of a total microsomal RNA without se- 
lective loss of actively-labelled components. 
The procedure is rapid. It is carried out at zero 
degrees and has been equally applicable to both 
RPC-20 tumor and liver, with yields of con- 
sistently between 87 to 100%. Furthermore, in 
this procedure de-proteinization with phenol is 
achieved with no loss in specific activity. 

By means of this technique, rapidly-labelled 
RNA was obtained from both RPC-20 tumors 
and livers of tumor-bearing animals that sedi- 
mented as a sharp 18S peak superimposed 



NATIONAL CANCER INSTITUTE 



11 



upon a heterogeneous size distribution ranging 
from lOS to more than SOS. The label did not 
appear in the sedimentation range commonly 
ascribed to mammalian, pulse-labelled "mes- 
senger RNA" between 4S and 18S, and it was 
only by exposure of the preparations to tem- 
peratures of 37-60° in low anionic strength 
that a displacement of the rapidly-sedimented, 
post-labelled RNA into the so-called "mes- 
senger" region of the gradients was obtained. 

With Dr. C. Abel of Field Studies, prelimi- 
nary study of the ability of RNA obtained in 
this manner to stimulate an amino acid incor- 
poration in the cell-free E. coli system of Ni- 
I'enberg has shown that "messenger activity" 
of the tumor RNA was at least partially pre- 
served during the preparative procedure. 

A subline of the RPC-20 ascites plasma cell 
tumor appears to have lost its capacity for 
synthesis of the characteristic RFC protein. 
This non-producing line has been examined by 
electron microscopy by Dr. R. Ziegel (Viral 
Oncology) and still retains the cytological ap- 
pearances of a plasma cell tumor. The growth 
of this line has greatly increased over that of 
the remaining protein-producing RPC-20 lines. 
Dr. Ziegel has found occasional C-type "viral" 
particles budding from the surface of the non- 
producing cell, a phenomenon not previously 
associated with the murine plasma cell tumors. 

Following intraperitoneal injection of as- 
cites plasma cell tumors, the malignant cells 
tend to implant on the mesenteric surfaces and 
form solid nodules (in addition to the free cell 
population) . When the mesentaries were exam- 
ined with a dissecting microscope at varying 
times after transplantation of free ascites tu- 
mor cells, discrete nodules were discernible 
after 7 days post-transplant but not at 5 days. 
Individual nodules were easily dissectable, and 
when transferred to antigen wells of Ouchter- 
lony plates and lysed with detergents, such 
nodules invariably liberated specific secretory 
protein in easily detectable amounts. Nodules 
from the non-producing ascites tumor lines 
gave no reaction with specific antisera. In the 
absence of detergent, nodules from the produc- 
ing RPC-20 line gave no reaction with the spe- 
cific antisera, indicating that the specific pro- 
tein responsible for precipitant arcs was 
contained within the cells and did not rep- 



resent contamination with ambiant secretory 
protein. 

Tumor-Host Relations Section 

The interaction between the tumor and the 
host organism is being investigated at several 
different levels of organization ranging from 
the whole animal down to the molecular level 
and makes use of a variety of biological sys- 
tems. 

Dr. P. Gullino, by the use of a "tissue isolat- 
ed" tumor preparation into which he has im- 
planted a chamber for obtaining interstitial 
fluid, has studied the environment of the neo- 
plastic cell and has found anomalous behavior 
in the tumor interstitial fluid as compared with 
other fluids of the host. As expected, adminis- 
tration of NaHCOa produced alkylosis in the 
blood lymph and subcutaneous interstitial fluid 
of the host, but acidosis in tumor interstitial 
fluid. The pH of this fluid normally about 0.3 
unit lower than that of the host fluids, became 
0.6 of a unit lower as a result of the bicarbon- 
ate treatment; the difference in bicarbonate in- 
creased from 3 to 5 mM per liter and the differ- 
ence in CO2 tension increased from 35 to 68 
mm Hg. These remarkable modifications could 
be obtained with a relatively mild treatment, 
that is the substitution of drinking water by 
0.1% of sodium bicarbonate solution. The dif- 
ference of 0.3 of a pH unit and 35 mm Hg in 
CO2 tension between the tumor and host inter- 
stitial fluid was also maintained during aci- 
dosis produced by ammonium chloride. Thus, 
an induced acidosis of pH 6.6 to pH 6.7 could 
be maintained in neoplastic tissue, a pH which 
is known to impair the survival of neoplastic 
cells in vitro. Inhalation of air containing 5 to 
10% CO2 produced an increase of the CO2 ten- 
sion of the tumor interstitial fluid to 200 mm 
Hg, about 100 mm Hg higher than that found 
in other body fluids of the host. These studies 
indicate that it is possible to produce a selec- 
tive effect in the interstitial fluid of a tumor 
and should have implications in the design of 
chemotherapeutic procedures. 

Since the tissue-isolated preparation allows 
sampling of both the afferent and efferent 
blood through the tumor as well as a measure- 
ment of blood flow, it has been possible to 



12 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



study the degree of correlation between respi- 
ration and glycolysis of neoplastic tissues in 
vivo and in vitro and to evaluate whether the 
data collected from in vitro studies can be ap- 
plied to neoplastic cell populations growing in 
vivo. Preliminary results indicate that the 
coefficient of oxygen utilization by the tumors 
studied range from 47 to 69% and the ability 
of neoplastic tissues to remove oxygen from 
the blood is equal to or higher than that of nor- 
mal organs. Eeduction of oxygen supply is par- 
tially compensated for by the tumor with an 
increase of blood flow but not by enhancing the 
oxygen-utilizing capacity. The overall con- 
sumption of oxygen by neoplastic tissue in vivo 
is influenced by a variety of factors and is diffi- 
cult to assess. In the series of rat tumors exam- 
ined, the values ranged from 0.5 to 8.0 
mM/lOOg/hr. The higher values indicate that 
the Qo2 in vivo may be equal to the Q02 that 
have been measured in vitro with surviving 
slices. Moderate hyperglycemia (200 mg/100 
ml plasma) enhanced the oxygen consumption 
of the tumor but high levels of glycemia re- 
duced the oxygen utilization, implying an in 
vivo demonstration of the Crabtree effect. 

At the cellular level Dr. R. E. Greenfield is 
studying single cell suspensions from the tis- 
sues of normal and tumor-bearing animals. It 
has been shown that the respiratory rate of par- 
enchymal liver cell suspensions at a pH of 
7.0-7.2 is inversely related both to the concen- 
tration of cellular glycogen and to the glucose 
concentration of the media. Since cells isolated 
in the livers of male animals have, in general, 
higher levels of cellular glycogen than those 
from the female, cells from the male had lower 
respiratory rates than those from the female. 
At a pH of 7.6-7.8, on the other hand, respira- 
tory rates had no relationship to either glyco- 
gen or glucose in the media. The respiratory 
rates of cells from the female at this pH were 
high, and indeed doubled those from male cells. 
Under culture conditions, cells from a regener- 
ating liver still showed mitotic activity after 
three days and again after the fifth and sixth 
day. 

In collaboration with Dr. S. Perry (Medicine 
Branch, NCI) Dr. R. E. Greenfield has been 
studying the fractionation of cell suspensions 
by gradient centrifugation. In cooperation with 



Mr. G. Judson (Guest Worker) a new centri- 
fuge of 50 ml capacity has been developed with 
a disc-shaped, batch-type head, and without 
vanes. The general distribution of the cells 
within the centrifuge head can be observed 
during the acceleration and deceleration. The 
centrifuge was found to be suitable for gra- 
dients for cells up to a density of 1.13; at 
higher densities viscosity introduced difficul- 
ties. Clumping was found to be one of the prin- 
cipal obstacles to the clean separation of cells 
but clumping could be decreased by the use of 
polyvinylpyrolidone which had been dialyzed 
for 24 hours or passed through Sephadex G-50 
to remove the smaller molecules. Red cells and 
cells from chronic meylogenous leukemia have 
been fractionated in gradients of polyvinylpy- 
rolidone both by differential and buoyant den- 
sity centrifugation. Red cells were found to 
have a buoyant density range of 1.08-1.13. 
Lymphocytes, myelocytes, and polymorphonu- 
clear leukocytes from chronic myelogenous 
leukemia cells had average densities of 1.03, 
1.04, and 1.055 respectively. 

Drs. R. I. Mishell (Post-doctorate Fellow) 
and R. E. Greenfield have been studying im- 
mune and malignant plasma cells in tissue cul- 
ture, with the aim of establishing an in vitro 
system which will enable normal mouse spleen 
cells to be immunologically-induced to differen- 
tiate, proliferate, and function and which will 
permit malignant plasma cells to grow and 
synthesize myeloma globulins. The general 
procedure has been to modify culture condi- 
tions, assessing the response as measured by a 
quantitative assay which determines the number 
of cells in the population which synthesize 
specific antibody. The assay used is a modifi- 
cation of the agar hemolytic plaque technique 
developed by Jerne. Studies of spleens re- 
moved from mice at progressively longer inter- 
vals following a single in vivo immunization 
indicate that very few plaques (antibody 
synthesizing cells) can be recognized until 72 
hours after immunization. A peak response is 
observed at 4 to 5 days after which the number 
of plaques formed declines rapidly. As a result 
of a number of modifications in culture condi- 
tions, it has been found that the exygen concen- 
tration appears to be quite critical since oxy- 



NATIONAL CANCER INSTITUTE 



13 



gen in excess of 20% is toxic to the develop- 
ment of plaque forming cells, but does not ad- 
versely affect plasma cells that have already dif- 
ferentiated. Low levels of oxygen (2%) is also 
inhibitory but by a clearly different mechanism 
in that more cells actually differentiate in 2 
percent than in 20 percent oxygen but they do 
not survive as well. Furthermore, the toxicity 
of low oxygen is observed with previously dif- 
ferentiated cells as well as newly differentiat- 
ing cells. The size of inoculum is also of ex- 
treme importance since inocula of 5 X lO'' 
cells per cc are quite effective whereas very 
little differentiation can be detected with 1 x 
10' or with 2 X 10* under the culture condi- 
tions employed. Fetal bovine serum seems to 
supply a factor or factors which are essential 
for differentiation in that 30% serum results 
in a 5-fold increase in the number of cells that 
differentiate as compared to 5% serum. 

Dr. E. Shelton has just returned from a year 
of study in the laboratories of Dr. W. Bern- 
hard, Laboratoire de Microscopie Electronique, 
Institut de Eecherches sur le Cancer, Villejuif, 
France and has established an electron micros- 
copy unit in the Laboratory of Biochemistry. 
During the year at Villejuif, a study was made 
of the effect of various fixing and embedding 
procedures on nucleohistone preparations. Of 
greatest interest were the results obtained 
when formalin-fixed material was embedded 
in 2-hydroxyethylmethylacrilate (GMA). GMA- 
embedded material showed no structure under 
the electron microscope other than a uniformly 
dense sheet. Upon treatment with pepsin or 
tris buffer however a network of thick fila- 
ments appeared. Tris buffer is known to ex- 
tract histones and although the protease pepsin 
does not act extensively on basic proteins, it 
appears to be quite effective on thin sections of 
formalin-fixed nucleohistone preparations. Sim- 
ilarly, when thin sections were treated only 
with DNase, filaments were again brought into 
evidence. When both pepsin and DNase were 
employed sequentially, all evidence of structure 
disappeared. These experiments suggest that 
conventional fixation and embedding proce- 
dures result in the loss of materials that are 
preserved by embedding in GMA. Selective en- 
zyme digestion of GMA-embedded materials 



reveals structures similar to those seen in the 
conventional preparations. 

Diffusion chamber studies on factors effect- 
ing the malignant transformation of tissues 
have been continued. Collaborative studies with 
Dr. B. Arneson of Boston failed to confirm the 
report of Levey and of Miller in which immu- 
nologic competence of neonatally thymecto- 
mized animals was restored by implantation of 
diffusion chambers containing newborn thjonus. 

The establishment of two tumor lines of the 
same origin differing markedly in catalase activ- 
ity without other apparent differences has 
provided an excellent system for the study of 
properties and interrelationships of cell popu- 
lations by Dr. M. Rechcigl, Jr. and Dr. R. E. 
Greenfield. These tumors have remained stable 
for over 70 generations of transplantation. 
When fragments of tumor from the high cata- 
lase line and the low catalase line were mixed 
together and injected subcutaneously, stable 
tumors of intermediate catalase values have 
been obtained for up to 30 generations. How- 
ever, on injection of fragments of the mixed 
tumor into the spleen, three-fourths of the 
metastases obtained in the liver had catalase 
values of 20-40 units whereas one-fourth of the 
metastases had catalase values of 90-140 units. 
Injection of the low catalase or high catalase 
tumor into the spleen gave only nodules with 
low (40 units) or high (200 units) levels of cat- 
alase, respectively. Whether the metastases 
from the mixed tumors with 90-140 units of cat- 
alase are still due to small mixed groups of 
cells or whether they are due to hybrid cells is 
not known. Of great interest is the observation 
that when small clumps of cells were prepared 
by the use of trypsin, PVP, and sucrose and 
were injected in different proportions of low 
and high catalase lines subcutaneously, the ini- 
tial tumors had catalase activity values approx- 
imating the calculated mean catalase level of 
the mixture. However, within the next two 
generations the catalase line except that in the 
third generation that mixture which consisted 
of 99 : 1 of low catalase line to high catalase line 
produced a tumor characteristic of the low 
catalase line. 

The physiologic role of catalase and related 
enzymes and the mechanisms of their regula- 
tion in normal and neoplastic tissues has been a 



14 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



continuing line of investigation by Dr. M. 
Rechcigl, Jr. Using procedures previously de- 
scribed which are capable of separating cata- 
lase synthesis and catalase destruction, the de- 
pression of catalase activity observed in rats 
following the feeding of cycad meal or its toxic 
component, cycasin has been examined. It was 
found that whereas the rates of destruction 
were approximately the same in normal and 
cycasin-fed rats, the rate of catalase synthesis 
is roughly 20 percent of that in the normal 
rats. The results are comparable to the earlier 
findings in this laboratory where the lower cat- 
alase activity observed during protein defi- 
ciency was entirely explained by the lower rate 
of catalase synthesis in protein deficient ani- 
mals. 

In collaboration with Dr. Z. Hruban of the 
University of Chicago several characteristic 
hepatomas of different growth rates and cata- 
lase content were examined by electron micros- 
copy and the enzyme contents of the tumors 
were measured. Microbodies were positively 
identified in the HC (high catalase), LC (low 
catalase) , Reuber H-35 and Morris 5123 hepa- 
tomas but were completely absent from fast- 
growing Novikoff and Morris 3683 hepatomas. 
Examination of the data indicated that only 
hepatomas with a relatively high catalase and 
uricase activity contain microbodies and the 
morphology of the microbodies were quite char- 
acteristic. For example, in the Reuber H-35 
hepatoma, the inclusions were large and con- 
tained large crystalloids whereas the 5123 
hepatoma had small microbodies and the ma- 
trix was electron dense. The LC and HC hepa- 
toma-microbodies were intermediate in size but 
were distinguishable on the basis of the small 
crystalloids which they contained. Good corre- 
lation has been found between the activity of 
uricase and the presence of microbody crystal- 
loids with the highly characteristic crystalloid 
structure being shown by electron microscopy 
to be identical with the structure of purified 
uricase crystals. 

The use of catalase as a genetic marker in 
C3H mice has been described previously. Re- 
cent surprising finding of a high catalase level 
in one of the C57BL substrains has prompted a 
thorough study by Dr. M. Reichcigl, Jr. Dr. W. 
E. Heston and Dr. H. A. Hoffman (Laboratory 



of Biology, NCI) of the liver and kidney cat- 
alase activity of various C57 strains and sub- 
strains and of strain C58 all of which were orig- 
inally derived from the Lathrop stock. No 
variation was found with respect to kidney 
catalase activity, however, the variation in liver 
catalase activity in certain substrains has sug- 
gested that a mutation resulting in a low liver 
catalase activity must have been present in the 
original Lathrop stock and that later reverse 
mutation to high level had occurred in two of 
the substrains. After a thorough genetic anal- 
ysis reported elsewhere, the difference in liver 
catalase activity was shown to be due to a 
single gene difference, surprisingly with the 
low catalase level of activity dominant over the 
high. This gene had no apparent effect on the 
level of kidney catalase activity. 

LABORATORY OF BIOLOGY 

During the year the Laboratory of Biology 
has maintained its usual high productivity in 
basic areas of research as they relate to cancer. 
A total of 30 original papers have been pub- 
lished by the various members of the staff. 
Areas of immunology, biochemical genetics, 
and cell physiology and nutrition have received 
special emphasis but some of the work has also 
been in areas of virology, endocrinology, mam- 
malian genetics, biochemistry, and tissue cul- 
ture instrumentation and methodology. 

The year has been marked by changes in per- 
sonnel and program emphasis. Some of the 
older staff members are dropping out and 
younger members with new interests are being 
added. Dr. Charles Nicoll has joined the staff 
to strengthen the area of endocrinology. Dr. 
Daryl Granner who is also interested in endo- 
crine problems and Dr. George Lyon who is in 
the area of biochemistry have been added to 
the Laboratory as Research Associates. Dr. 
Melvin Reuber, a pathologist, is being trans- 
ferred into the Laboratory to strengthen the 
study of liver carcinogenesis — particularly of 
the spontaneous hepatomas of the mouse. Dr. 
Eva Leckband has joined Dr. Law's staff as an 
NIH fellow in his work on the role of the 
thymus in immunologic response. Dr. Ettore 
Appella, a protein chemist, is expected to join 
Dr. Potter on September 1, in their very ener- 



NATIONAL CANCER INSTITUTE 



15 



getic program on the differentiation of mye- 
loma immunoglobulins. We hope to bring in 
Dr. William Hall, an electron microscopist, on 
September 1 to fill this critical need of the La- 
boratory. Dr. Ram Parshad, a cytogeneticist, is 
joining the Tissue Culture Section in October 
as an International Fellow. In addition, three 
new Research Associates are joining the staff 
on July 1. While this spirit of youth taxes the 
budget it certainly envigorates the research 
program. 

One of the greatest honors for the Labora- 
tory this year was for Dr. Law to have been 
chosen to give the Clowes Memorial Lecture, 
thus recognizing him as one of the most out- 
standing investigators in cancer research. 

The following summary includes studies cho- 
sen to illustrate the program of the Labora- 
tory. 

Immunology 

Dr. Law has made a number of new observa- 
tions in his study of the role of the thymus in 
immunogenic response. After neonatal thymec- 
tomy NIH (Swiss-Webster) mice do not show 
signs of the "wasting" syndrome found in 
other strains. A survey for a number of contam- 
inating viruses showed no differences be- 
tween NIH strain and the other strains that do 
"waste". However, in his study of the effect of 
neonatal thymectomy of germ free mice, Dr. 
Mclntire showed the necessity of infection for 
the wasting syndrome. 

Law has shown that the humoral-thymic fac- 
tor for immune response is not species-specific 
because grafts of Osborne-Mendel rat thymic 
tissue can restore the immune response in 
neonatally thymectomized (BL x C3H) Fi mice. 

The thymus is thus linked to oncogenesis. 
Law has shown that neonatal thymectomy 
strikingly increases the sensitivity of C57BL 
and C3H/Lw mice to the oncogenic potential of 
several strains of polyoma virus. Resistance 
can be restored with thymic grafts and inocula- 
tion of spleen and lymph node cells but not of 
spleen cells from thymectomized mice. The ex- 
istence of an immunologic mechanism of the 
homograft type responsible for the resistance 
to polyoma virus oncogenesis is indicated by 
the work of Law and Ting. However, there was 



no evidence of such resistance to the mammary 
tumor virus or to methylcholanthrene carcino- 
genesis for thymectomized C3H mice had 
fewer mammary tumors than controls and 
were not more susceptible to the induction of 
fibrosarcomas with MCA. The effect of thymec- 
tomy on oncogenesis by other viruses and in 
other species is being investigated. 

Dr. Ting has demonstrated maternal trans- 
mission of antibody against polyoma virus. The 
milk was the route of transmission. He has also 
demonstrated transplantation resistance to Mo- 
loney lymphoma induced by two other murine 
leukemia viruses, PLLV and L-180 isolated in 
his laboratory. His results indicate that \ym- 
phoma induced by these three strains of virus 
share a common antigen. 

Miss Uphoff has continued her work on the 
immune response, using radiation to study the 
host-versus-graft reaction and the graft- 
versus-graft reaction. She has also demonstrat- 
ed genetic factors that influence irradiation 
protection by bone marrow. 

Virology 

Sanford has been trying to induce the malig- 
nant change in cells in vitro with Polyoma 
virus but whereas the virus infected cells ap- 
peared to transform earlier, the control cells 
also transformed. However, in the virus infect- 
ed cells the transformation occurred long after 
the virus infection. Cell morphologic changes 
occurred but could not be correlated with any- 
thing except cells that were treated with 
high multiplicities of virus showed a delayed 
morphologic response. She also tested 5 strains 
of mouse and hamster cells for their capacity 
to develop polyoma-induced transplantation an- 
tigens in vitro. Such antigens were induced but 
their formation appeared to be a separate, in- 
dependent phenomenon from the neoplastic 
change. Dalton has observed C particles in sev- 
eral of Sanford's cell clones in vitro but they 
differed from mouse leukemia viruses and pro- 
duced no lesions Avhen assayed in newborn mice 
of several strains. When the clones were adopt- 
ed to the chemically defined media the C par- 
ticles disappeared. 

Dr. Ting has demonstrated the presence of 
viral genes in virus-free polyoma tumor cells. 



16 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Polyoma tumor 4198 originally induced in C3H 
with polyoma virus but now with no demon- 
strable virus was superinfected with SE 3049 
virus. SE 3049 virus that had been grown on 
4198 could then protect C3H mice against 4198 
transplants although SE 8049 grovim on nor- 
mal mouse embryo cells gave only weak protec- 
tion. 

Dr. Law is continuing his study of congeni- 
tal transmission of a leukemogenic virus, MLV. 
Three strains, C3Hf/Gs, C3Hf/Lw, and EFM 
have now been maintained through 15 genera- 
tions as high leukemic strains owing to the 
transfer of the MLV through the mother's 
milk. Occasionally however, sublines will re- 
vert to low-leukemic lines because of failure of 
the MLV to be transmitted. There is no evi- 
dence of contact infectivity of MLV. 

Dr. Andervont has continued his studies of 
the mammary tumor virus. In strain RIII he 
has noted that its effectiveness was decreased 
in certain females and did not regain its activ- 
ity in many subsequent generations of descen- 
dants. When C3H mammary tumor virus is put 
in RIII mice it can also lose its effectiveness in 
certain females. When the RIII mammary tu- 
mor virus is introduced into C3H it does not 
increase in potency. 

Dr. Barrett, in collaboration with Dr. 
Deringer, has been studying the latency of the 
mammary tumor virus to see if it represents a 
period of maturation of the virus-host relation 
or is primarily a maturation of the tissue sus- 
ceptibility. He observed that the length of the 
latency period was the same whether the virus 
was injected into newborn test females, those 
one month old, or those 10 months old which 
indicates that latency represents a maturation 
of host-virus relationship. 

Biochemistry 

The discovery of feedback control over the 
uridine kinase reaction by Dr. Anderson and 
collaborators constitutes the first demonstra- 
tion of such control over a salvage pathway for 
pyrimidine ribonucleotide biosynthesis. The 
control is very effectively exerted by the end- 
products CTP and UTP, and the mechanism of 
the inhibition has proved to be exceedingly in- 
teresting. Feedback control of the kinase has 



been found in a variety of cell types. However, 
in microorganisms that utilize uracil, a similar 
kind of feedback control is exerted, instead, on 
UMP pyrophosphorylase. The pyrophosphory- 
lase is probably the major salvage pathway of 
biosynthesis in these cells, whereas the kinase 
pathway predominates in other organisms, in- 
cluding the mammalian cell systems. Studies 
(mostly by Dr. Lyon, a research associate here 
since July 1964) are also being made on uri- 
dine phosphorylase, using preparations from 
both microorganisms and mammalian tissues; 
the studies are aimed at defining whether this 
enzyme serves an anabolic or catabolic role in 
metabolism and whether this role determines 
the properties of the enzyme, including its 
substrate specificity and response to control 
mechanisms. In connection with our studies on 
the sites of action of antimetabolites, careful 
and extensive work has been done (by Mr. 
Ciardi) on the identification of various purine 
and pyrimidine metabolites. These have in- 
cluded pioneer use of the new and versatile 
method of thin-layer chromatography. 

Biochemical Genetics 

Dr. Potter who recently has been collaborat- 
ing with Dr. Appella has continued his very ac- 
tive study of myeloma immunoglobulins in 
BALB/c mice with plasma cell tumors. The 
transplantable plasma cell tumor is like a clone 
of cells in respect to protein secretion and 
BALB/c mice with these tumors have large 
quantities of specific myeloma immunoglobu- 
lins in their serum and urine. This is the best 
means of separating in relatively pure foi'm 
specific immunoglobulins from the heteroge- 
neous family of immunoglobulin molecules that 
exist in the normal individual. The light and 
heavy polypeptide chain subunits isolated from 
a series of myeloma proteins were classified by 
antigenic structure, tryptic peptide maps, and 
amino acid composition. It was found that 
there were two types of light chains, kappa and 
lambda, and five types of heavy chains, alpha, 
mu, gamma F, gammaGBe-1, and gamma- 
GBe-2. Thus seven structural genes which 
participate in immunoglobulin formation in the 
inbred BALB/c mouse have been identified. 
When the heavy chains from an individual 



NATIONAL CANCER INSTITUTE 



17 



class for example gamma F were compared it 
was found that there were differences in the 
amino acid and tryptic peptide map composi- 
tion. Similar differences were found among 
gammaGBe-1, gammaGBe-2, and gamma A 
heavy chains. These findings have implications 
in the mechanism of the chemical basis of anti- 
body variation. 

Potter has studied the genetic control of 
heavy chain polypeptide synthesis in collabora- 
tion with R. Lieberman and S. Dray of NIAID. 
Utilizing myeloma proteins and allotypic preci- 
pitating antibodies prepared by immunizing 
mice of one strain with immunoglobulins of an- 
other it has been shown that mice of the a4 al- 
lotype group differ from the al BALB/c in the 
gammaGBe-1 heavy chain. Genetic linkage of 
the gammaGBe-1 and gammaGBe-2 heavy 
chain specificities has been demonstrated. 

Dr. Mclntire in studying the Bence Jones 
proteins has demonstrated that a group of 
them not previously known to function as a 
light chain are definitely found as the light 
chain of a myeloma macroglobulin and are also 
found by antigenic testing in the macroglobulin 
of normal mice. This chain now designated as 
the kappa chain is found in immunoglobulins 
other than macroglobulin in strains of mice 
other than BALB/c. This means there are at 
least two antigenic types of light chains in 
mouse immunoglobulins, the other being 
lambda or the L chain, which corresponds to 
the number of types for human immunoglobu- 
lins. Special study with Kuff of a macroglobu- 
lin produced by one of his plasma cell tumors 
indicated that the macroglobulin, like other 
myeloma globulins of mice, is composed of 2 
different size polypeptide chains (heavy and 
light) linked by disulfide bond. 

In studies of starch gel electrophoresis pat- 
terns of urine of male and female mice of 18 
inbred strains. Dr. Hoffman has found 6 differ- 
ent electrophoretic patterns, two different 
3-band patterns, and four different 4-band pat- 
terns. From genetic analyses the 3- or 4-band 
electrophoretic mouse urinary protein types 
appear to be under the control of a pair of co- 
dominant alleles. However, the locus for the 
MUP types appears to be complex with at least 
6 alleles represented in the 18 inbred strains. 



Hoffman has also been studying serum pro- 
tein variants or serotypes. A serum protein has 
been discovered in BALB/c that is antigenic to 
the NBL strain. From immunoelectrophoretic 
studies it appears that the protein is probably 
a beta-globulin with a high molecular weight. 
Genetic linkage studies of this protein with the 
immunoglobulin Ig-1 trait, the low liver cata- 
lase Ce trait, and a variant of caracul coat gene 
Ca are in progress. 

Mammalian Genetics 

In their studies of effects of specific genes on 
occurrence of tumors, Heston and Vlahakis 
have demonstrated that the newly discovered 
mutation viable yellow A^ greatly increases 
susceptibility to mammary tumors. Like its al- 
lele, lethal yellow, it also increases occurrence 
of pulmonary tumors and hepatomas. A"^ arose 
in strain C3H and the sublime with this gene, 
C3H-A'''', is now the most susceptible strain we 
have to mammary tumors and hepatomas. In 
contrast, the dwarf gene that stops growth at 7 
to 10 grams completely inhibits mammary tu- 
mors, hepatomas, and urethan induced lung tu- 
mors except for a few very small lung tumors 
that do not grow. 

The very high incidence of hepatomas in 
strain C3H, especially C3H-A^ and their hy- 
brids, is of special significance. In CSH-A"^ 
males hepatomas occur in 100 percent of the 
animals and each animal has many hepatomas. 
This now provides excellent material for 
further study of factors in the etiology of this 
neoplasm. 

Dr. Deringer has reported an incidence of 22 
percent of mammary tumors in her BALB/c 
females in contrast to the extremely low inci- 
dence reported by others. This is probably ow- 
ing to her maintaining them in a healthy condi- 
tion to a much greater age and observing them 
more closely. She also noted 10 percent with 
reticular neoplasms. This is of significance 
since BALB/c is used so extensively as a test 
strain. She has also recorded the occurrence of 
amyloid in organs of strain BL/LyDe mice. 
This would be an excellent strain for studying 
amyloidosis. Deringer has reported an increase 
in reticular neoplasms in her DBA/2eB mice 



18 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



with urethan. She had also reported an in- 
crease in hemangioendotheliomas and has now 
shown that these are transplantable. 

Endocrinology 

Dr. Nicoll joined the laboratory September 
1, 1964 and has now gotten underway a rather 
extensive endocrinology program including 
growth of the mammary gland and the role of 
the stroma which he has demonstrated to have 
an important regulatory action; in collabora- 
tion with Hoffman comparison of secretory ac- 
tion of normal and neoplastic mouse pituitary 
gland using organ culture and subjecting the 
medium to starch-gel electrophoresis ; an evolu- 
tionary study of prolactin; and the develop- 
ment and modification of the pigeon crop-sac 
assay of prolactin. During the year he has also 
published several papers on work done pre- 
viously. 

Dr. Granner, who joined the Laboratory in 
July 1964, is making a descriptive study of the 
premalignant mammary plaques in strain DD 
mice. He is also studying the effect of proges- 
terone as well as estrogen in induction of mam- 
mary tumors in strain A mice. 

Cell Physiology and Nutrition 

Dr. Evans and co-workers have adapted a 
number of additional cell lines including C3H 
mouse kidney, C3H mouse embryo, and green 
monkey kidney cells to chemically defined me- 
dia and this media supplemented with horse 
serum and with calf serum. On the basis of 
growth of intraocular transplants the time of 
the malignant transformation is fixed with re- 
markable biological reproducibility. One of the 
most outstanding findings is that cells in the 
media with calf serum transform much later 
(if at all) than those in the media with horse 
serum. Studies are underway to try to deter- 
mine what this difference between horse serum 
and calf serum in producing the malignant 
transformation is due to. The foetal calf serum 
delays but does not arrest alterations in chro- 
mosomes. 

For the first time non-malignant freshly ex- 
planted mouse cells have been grown in chemi- 
cally defined media in large enough quantities 
and good enough quality to be used in nutri- 



tional and transformation studies. Dr. Evans 
has now shown that C3H mouse embryo cells 
explanted directly into the chemically defined 
media with absolutely no added serum or any 
supplement will undergo the malignant trans- 
formation in approximately 130 days. A con- 
siderable list of biochemical and nutritional 
differences in cells in vitro have now been 
recorded by the tissue culture group. 

Evans has shown that HeLa, H.Ep.2, a 
hamster cell strain, and one mouse embryo cell 
strain all require both vitamin B12 and i-inosi- 
tol. The liver I strain may require both vita- 
mins A and D. The green monkey kidney and 
liver n strains appear to be less specific than 
the other strains in requirements. 

Sanford had noted consistently higher up- 
take of glucose and production of lactate in a 
high-tumor producing clone of cells than in a 
low-tumor producing clone. However, inoculum 
size was variable and when its effect was tested 
it was found that higher uptake of glucose and 
production of lactate occurred in clones grown 
from low inoculum size than from high. 

Dr. Westfall has made a number of observa- 
tions of five enzymes in cells on chemical 
defined media. For example, cells of two inde- 
pendent adaptations of a clone of mouse liver 
cells to chemically defined media showed differ- 
ent arginase activities. One continued to show 
high arginase of the progenitor line while that 
in the other dropped. In another study he noted 
that cells grown in media supplemented with 
calf serum had twice to 3 times the lactate de- 
hydrogenase as those in media supplemented 
with horse serum. These biochemical markers 
are useful in characterizing differences cells 
undergo in long-term cultivation in vitro. 

Sanford had made the interesting observa- 
tion that when she clones cells of Swarm's tera- 
toma they lose their ability to differentiate. 
This was noted in 7 clones, whereas parental 
lines not cloned maintained this ability. This 
seems to answer a question that has been asked 
of teratoma cells for many years. 

In cooperation with Tjio and Wang, Sanford 
has been trying to hybridize cells in culture. 
Using chromosomal markers they as yet have 
no evidence of hybridization and thus are not 
able to confirm the work of Ephrussi and 
others. 



NATIONAL CANCER INSTITUTE 



19 



Tissue Culture Methodology 

Dr. Bryant has made further studies on the 
shaker flasks for cultures. He now has H.Ep.2 
and mast cell 815 in chemically defined media 
in the shaker flasks. The larger the flask the 
lower the concentration of cells and the stress 
on the cells in the mediimi sized flask is not 
enough more than the smaller flask to prevent 
their use. Therefore the medium flask is the 
choice for building up high cell populations 
suitable for the continuous flow culture. This 
has been done with line 2071 cells. 

Mr. Andresen is being successful with the 
mass stirrer flask. This permits continuous nu- 
trient fluid renewal and continuous withdrawal 
of cells. This is the only place this can be done. 
Commercial organizations still have to use the 
batch system. 

Both Evans and Sanford have been testing 
several commercial media qualitatively and 
quantitatively but none compares favorably 
with their own media. 

The motion picture camera is being used suc- 
cessfully to determine growth and mitotic rates 
in cells in the chemically defined media. 

PATHOLOGIC ANATOMY BRANCH 

In addition to the diagnostic pathology serv- 
ices performed in the Department of Patho- 
logic Anatomy, the permanent staff and resi- 
dents study and make reports on human case 
material and perform a variety of types of re- 
search. Most of the specific animal and labora- 
tory research projects of the staff have been 
mentioned earlier in this summary, so the prin- 
cipal items listed here will be those performed 
by the resident staff. Many of the studies of 
human material are in collaboration with clini- 
cal staff or with staff of the pathology depart- 
ment. 

Dr. Berard published a number of articles 
this year on experimental work he performed 
at Walter Reed Army Institute of Research. 
These included studies on the effect of ascorbic 
acid and deuterium oxide on wound healing. In 
addition he has studied a number of diseases in 
man including: clostridial septicemia in pa- 
tients with cancer, disseminated histiocytosis, 
the central nervous system in patients with 
congenital heart disease, the hypothalamus in a 



patient with acute intermittent porphyria, a 
defect of antidiuretic hormone activity, ocular 
pathology in patients with leukemia, coarcta- 
tion of the aorta with ventricular septal defect. 
In addition he is collaborating with Dr. Gittes 
on the effects of pineal extract on the testis of 
rats and with Dr. Malmgren on immunologic 
responses in tumor-bearing mice. 

Dr. Chambers has studied patients with Sjo- 
gren's disease, alopecia and a malignant lym- 
phoma and has been attempting to develop a 
line of African Ijonphoma cells adapted to 
Spinner culture. In addition he is collaborating 
with Dr. Gelderman in studying the effect of 
EDTA and mitomycin on experimental mela- 
noma in tissue culture. 

Dr. Gelderman has published several articles 
on LDH isozymes this year. One of these is as a 
chapter in a book by Sunderman and Sunder- 
man, The Serum Proteins and the Dysproteine- 
mias. In addition he is doing several experi- 
ments with EDTA and mitomycin designed to 
elucidate the molecular basis for cancer ther- 
apy. He is studying cases of fibrocystic disease 
of the pancreas and of the cerebellar changes 
in patients with leukemia. 

Dr. Grimley has continued developing tech- 
niques for using light microscopy and electron 
microscopy on the same tissue. He is also 
studying human cases of cynomegalic inclusion 
disease, carotid body tumor, and the associa- 
tion of glioblastoma with other tumors. 

Dr. MacLowry has been studying the juxta- 
glomerular apparatus in experimental hyper- 
tension and spleno-renul shunt in dogs. In addi- 
tion he is studying patients with spermatogenic 
arrest following chemotherapy and is helping 
establish a data retrieval program for the 
Surgery Branch. He also has reviewed the 
lungs of mice inoculated with T241 sarcoma 
and with ethiodol and poppy seed oil. 

Dr. Wright with Dr. Grimley published a 
paper on Torulopsis glabrata infection in man. 
He is currently working on a number of human 
diseases, including congenital anomalies of the 
entire autopsy population, cerebellar changes 
in patients with leukemia, conduction defects 
in patients with calcific aortic stenosis and my- 
cosis fungoides. He also is working with Dr. 
Thomas on a study of immunity in mice inocu- 
lated with L1210 leukemia. 



20 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



LABORATORY OF PATHOLOGY 

Introduction 

The work in pathology falls into two general 
categories: the work in the Laboratory of 
Pathology and the work in the Department of 
Pathologic Anatomy. The staff of Pathologic 
Anatomy performs the autopsies, examines the 
biopsies and surgical pathology specimens and 
does exfoliative cytology for patients in the 
Clinical Center. 

The primary aim of those in the Laboratory 
of Pathology is to carry out experimental 
cancer research and for this work they use a 
variety of techniques and experimental ani- 
mals. Each member carries out his own experi- 
ments, two or more members collaborate with 
each other and individual members collaborate 
with others in the National Cancer Institute. 

There is free exchange of information and 
help between and among all the pathologists. 
While the Annual Report reflects in general the 
work of the two groups in pathology, it also 
shows that there is considerable interchange, 
and interchange is constantly in progress. Most 
of the members of the Department of Patho- 
logic Anatomy have under study one or more 
problems involving cancer in man or some dis- 
ease in man which they carry out either inde- 
pendently or in collaboration with the clini- 
cians. The autopsies and the surgical pathology 
specimens and biopsies are examined with the 
greatest of care. They are worked up by resi- 
dents and their diagnoses are all reviewed by 
senior staff members. 

A notable accomplishment contributed by the 
head of the Department of Pathologic Anat- 
omy, Dr. Louis B. Thomas, is his work on a 
committee of the College of American Patholo- 
gists which has devised a nomenclature and 
code for pathological lesions. By the use of this 
the pathological material from the human cases 
have been arranged for data processing, and it 
is now in operation. 

Most of the members of the Department of 
Pathologic Anatomy also carry out experimen- 
tal research using animals, tissue culture and 
other special techniques. The residents in Path- 
ologic Anatomy are particularly ambitious in 
carrying out research programs. 



Experimental 

The experimental work in the Laboratory of 
Pathology is not restricted to a single project, 
or to a group of closely related projects, but 
each pathologist follows his particular line of 
interest and training. Because of this diversity 
the projects in the laboratory have been sepa- 
rated into 5 rather loose categories for conven- 
ience in preparing this summary. These will 
be: 

Cancer in Man, and Related Animal Studies 

A. Geographic Pathology of Cancer 

B. Exfoliative Cytology 

C. Possible Etiologic Factors in Human 
Environment 

Cancer in Animals 

A. Pathogenesis 

B. Modifications in Carcinogenesis 

C. Biologic Factors in Neoplasia 

D. Development of Spontaneous Tumors 

Viral Carcinogenesis. Problems of immunology 
and resistance. 

A. Polyoma Virus 

B. SV40 and Adenovirus 

C. Leukemogenic Viruses 

D. Immunology and Resistance 

Accumulation of Data. Laboratory animals and 
other species. 

A. Information on Laboratory Animals 

B. Phylo genetic Aspects of Neoplasia 

Collaborative Research 

A. Methods 

B. Examples 

Cancer in Man and Related Animal 
Studies 

Since all but one of the members of the Pro- 
fessional Staff of the Laboratory of Pathology 
and Department of Pathologic Anatomy are 
medically trained, many research activities are 
correlated with the problem of human cancer. 
Several projects are directly utilizing human 
material, and others are testing in animals 
substances in the human environment that may 
be carcinogenic. 

A. Geographic Pathology of Cancer. — Infor- 
mation is accumulating that certain forms of 
cancer in human population groups occur in a 



r 



NATIONAL CANCER INSTITUTE 



21 



higher incidence than would be expected. Inten- 
sive study of such groups, and the recognition 
of possible etiologic agents which can be tested 
on experimental animals may identify some en- 
vironmental factors which could be eliminated 
or controlled and thereby prevent many cases 
of human cancer. The following studies have 
this aim: 

1. Maps of different geographic areas are in 
preparation to show the relative frequency of 
different types of cancer. When it is recognized 
that some tj^pe is especially common, a more 
intensive study of the population group is indi- 
cated. (Dunham and Bailar) 

2. The following types of cancer are being 
investigated in particular geographic areas : 

a. Bladder cancer in New Orleans. White 
males over 60 in this city are frequently affect- 
ed. All histologic sections have been reviewed 
by the same pathologists (Stewart and Rab- 
son) to establish uniformity in diagnosis. 
Questionnaires on past history and possible ex- 
posure to carcinogens are being analyzed. 
(Dunham) 

b. Uterine cancer in New York City, Israel 
and Washington, D.C. This type of cancer is 
notably frequent in Negro women and infre- 
quent in Jewish women. Histologic sections 
have been examined from patients with cancer, 
and over 3500 women interviewed. The data 
will be coded in an effort to detect an environ- 
mental factor that may account for the racial 
difference. (Stewart, Dunham, Thomas, Edg- 
comb) . A survey of uterine cervical cancer in 
Colombia, S.A. where the incidence appears to 
be high, is in progress. (Malmgren) (See next 
page) . 

c. Lung cancer in Veterans of World War I. 
The medical history, and biopsy and autopsy 
material from this group is generally acces- 
sible. Histological material has all been re- 
viewed by the same pathologist, and a uniform 
classification of histologic types has been made. 
This survey indicated the unreliability of ran- 
dom histologic diagnoses which have often 
been accepted uncritically by statisticians or 
epidemiologists. All available cases have now 
been reviewed, and the results are being coded. 
This study will supply important information 
on the influence of smoking on the incidence of 
lung cancer within this group. (Herrold) 



d. Gastric Cancer in Japan. Cancer of the 
stomach is remarkably frequent in several un- 
related human populations, and in Japan, this 
type of cancer is especially common. Migrant 
Japanese in Hawaii offer a good group of genet- 
ically similar people in a different geographic 
area for comparison. The histologic sections 
are being reviewed by the same pathologist 
(Herrold), and any association with gastric 
ulcer, polyps or intestinal metaplasia is noted. 
A correlation will be made with data collected 
by a team of epidemiologists. An investigation 
is also made for viruses (Bryan) ; and electron 
microscopy and histochemical information is 
obtained when possible. This is a long term 
study that will require many years, but a pre- 
liminary report wll be given at the Interna- 
tional Congress in J apan in 1966. (Herrold, 
Stewart, pathologists from Japan) 

e. Malignant lymphoma in the U.S. states 
and Africa. The frequency of this form of 
cancer in children in Africa has aroused much 
speculation as to possible etiologic factors. It 
became important to determine whether this 
malignant lymphoma of unusual histologic type 
and anatomical distribution was restricted to 
Africa or whether a similar neoplasm occurred 
in other geographic areas. Histologic study of 
cases in the U.S. revealed that I/3 of the U.S. 
cases wei*e of similar histologic type, but the 
disease is rare in comparison with Africa and 
manifestation in the jaw is infrequent. In the 
U.S. the relative numbers of the specific histo- 
logic types of malignant lymphomas were 
found to differ in children and in young adults. 
The conclusion was reached that the tumor in 
Africa is an "incidence" phenomenon, and not 
a unique type of neoplasm. (O'Conor, NCI, 
Smith, AFIP, and Rappaport from University 
of Chicago) 

Additional studies were made on a case 
brought from Africa to the Clinical Center. No 
particles were revealed on E.M. study of the 
original tumor, but particles were found after 
several transfers in tissue culture. This con- 
firmed other work performed with tissue from 
this case at the National Cancer Institute. (O'- 
Conor, Rabson) 

B. Exfoliative Cytology. — A diagnostic serv- 
ice in exfoliative cytology is supplied to the 
Clinical Center, but in addition to this, a 



22 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



number of research studies are in progress in 
collaboration with various clinical services. 
These relate to the presence of neoplastic or 
cancer patients is not closely correlated with 
other cells on body surfaces or in fluids, and 
sex chromatin in exfoliated cells. It has been 
determined: 

1. Number of neoplastic cells in the blood of 
metastasis, but a possible increase of mega- 
karyocytes in blood in patients with cancer is 
found. 

2. Cancer cells are often recovered from 
wound washings after cancer surgery, but time 
will be required to determine whether the fre- 
quency is correlated with recurrence in the 
wound site. 

3. Leukemic cells in the spinal fluid provide 
a reliable indication of central nervous system 
involvement and assessment of therapy. 

A comparison of cervical cancer in Connecti- 
cut and Southwest Region of England has been 
reported. (Thomas and Bailar) Additional 
reports from this survey will include a correla- 
tion of 5-year survival rates with the histologi- 
cal features of the cancer found in the two 
areas and a detailed description of the cancers 
found among women in the two areas. 

4. An improved technique for identification 
of sex-chromain in cells from the buccal mu- 
cosa has been developed, and it appears that 
there is a correlation between the hormonal 
status in the female and the sex chromatin 
count, since a decrease in sex chromatin was 
found at the time of ovulation. 

5. The reported presence of a "malignant as- 
sociated change" in cells of the buccal mucosa 
was not confirmed. 

6. A technique for identifying cells from me- 
sothelial surfaces by E.M. study is being 
prepared. 

7. A method for coding findings in exfolia- 
tive cytology is being prepared. (Malmgren) 

C. Possible Etiologie Factors in Human En- 
vironment. — 1. Adsorbates from drinking 
water from New Orleans, where bladder cancer 
is high, and Birmingham, where it is low, are 
concentrated and given to mice. Betel quid 
ingredients and tobacco snuff are being tested 
in the cheek pouch of hamsters. DMSO (di- 
methyl sulfoxide), a chemical long used as a 
commercial solvent and lately proposed as a 



therapeutic agent because it readily permeates 
the skin, is being investigated as a possible car- 
cinogen in mice. No results can yet be reported 
from these experiments because the effect of 
weak carcinogens such as some of these may be 
difficult to assess, and requires a long period. 
(Dunham) 

2. A study on the effect of two strains of 
Schistosoma hoematobium. Gold Coast and 
Egyptian, on the hamster are continuing. This 
organism is associated with bladder cancer in 
Egypt but not in Africa. Histologic study is not 
yet completed. (Thomas, Berry). 

3. Thorium dioxide, once used as an x-ray 
contrast medium, has produced vascular neo- 
plasms of the spleen in guinea pigs two years 
after administration. It is hoped that a dose 
effect demonstrated may be studied in greater 
detail in guinea pigs. (Swarm) 

4. Numerous other substances to which hu- 
man beings are exposed have been tested, or 
are now being tested but the results so far are 
negative or incomplete. Material from an ochre 
barrel of African natives with a high incidence 
of carcinoma of the esophagus produced no 
cancer, and dihydrosaffarol (DHSF) a food 
coloring is negative so far. (O'Gara). Experi- 
ments with metals such as zinc, copper, iron, 
and heated fats given to rats are incomplete. 
(Hueper, O'Gara). Possible carcinogenicity of 
Cycad meal given to mice is incomplete but 
degenerative changes have been found in the 
liver. (O'Gara). Although a procedure effective 
in demonstrating pulmonary carcinogenesis 
from known chemical carcinogens was used, 
beryllium oxide, chromium oxide, asbestos, air 
pollutants and tobacco tar, trapped in inf arcted 
areas of the lung failed to produce neoplasms 
in rats. (Stanton). A number of substances 
mostly hormones were injected into newborn 
mice, but so far only estrogen proved to be car- 
cinogenic, and produced cervical cancer after 
two years. Progesterone produced no alteration 
but enovid given to newborns has caused le- 
sions in the uterine cervis 15 months later that 
may be preneoplastic. (Dunn) 

5. Possible relationships of cancer in man 
with acanthosis nigricans. A tissue culture line 
has been established from a uterine cancer in a 
human being who also had acanthosis nigri- 



NATIONAL CANCER INSTITUTE 



23 



cans. Cells from the culture were transplanted 
to the hamster cheek pouch. No melanocyte 
stimulating hormone was found. PPLO found 
earlier in the cell cultures was apparently a con- 
tamination of the test bacteriological cultures. 
The line is being continued, but no further 
work is now contemplated. (Dawe) (See #6) 

6. Pulmonary changes in patients with 
cancer. Dr. Powell has developed a machine to 
inflate human lungs under conditions of con- 
stant pressure and flow. He is using this to 
make a detailed study of pulmonary changes in 
patients with leukemia and of patients with 
emphysema. 

Cancer in Animals 

Many of the studies of cancer in animals are 
concerned with: A) Pathogenesis, or the steps 
by which cancer develops after exposure to a 
carcinogen. B) Modifications in the activity of 
carcinogens produced by altered conditions or 
substances. C) Biologic factors of neoplasia 
such as the bahavior of tumors during trans- 
plantation, metastasis, and effects of irradia- 
tion, and D) Spontaneous, transplanted and un- 
usual tumors. 

A. Pathogenesis. — 1. Development of tu- 
mors produced by 2-7-FAA when given to 
pregnant, and lactating mothers, to newborns, 
and to adult rats is being studied. The distribu- 
tion of skin appendage tumors produced by 
this chemical is being plotted on maps of the 
skin surface and a paper on histogenesis is be- 
ing prepared. The effect on young rats requires 
further time to determine. (Stewart and Snell) 

2. An intensive study is in progress on the 
histogenesis of liver cancer produced by N-2- 
fluorenyldiacetamide. The carcinogen is given 
at various dose levels. Early hyperplastic le- 
sions failed to grow when transplanted to the 
liver, but later ones grew and retained their 
histologic appearance. A histologic study is also 
being made of hepatomas in hybrid and inbred 
mice with the yellow gene. This detailed study 
of a single form of cancer may identify more 
precisely the point at which the neoplastic al- 
teration occurs. (Reuber) 

3. Nitrosamines were found to be potent car- 
cinogens, and were reported to produce brain 
tumors in rats. Nitrosamine compounds given 



by a variety of routes in hamsters produced tu- 
mors at similar sites, indicating that the distri- 
bution, metabolic pathways, and excretion of 
the carcinogen were the critical factors. Tu- 
mors of the olfactory neuroepithelium in Syr- 
ian hamsters were of especial interest, be- 
cause in previous reports these may have been 
mistaken for brain tumors by others. These 
studies emphasize that carcinogens may reach 
remote sites, and that when the site of tumor 
formation is in the lung, the carcinogen did not 
necessarily reach the lung by inhalation. (Her- 
rold) 

4. Syrian hamsters were given benzopyrene 
by intratracheal instillation and early changes 
in the bronchial epithelium were described. No 
tumors resulted from atmospheric pollutants 
or tobacco tar. An unanticipated finding from 
this experiment was the development of cryp- 
tococcus neoformans meningitis in one 
hamster. This disease occurs in man, where it 
has been presumed that the organism is blood- 
borne from a focus in the lung. Observations 
with the hamster suggest that the primary 
focus may be the nasal cavity and sinuses, 
from which sites the infection extends to the 
meninges. (Herrold) 

B. Modifications in Carcinogenesis. — 1. The 
effect of a carcinogen may be altered by the age 
of the host when it is given. Newborns are 
especially susceptible and some carcinogens 
pass the placental barrier. Nitrosamines given 
to pregnant rats caused tumors in the offspring. 
The monkey is not inherently refractory to 
carcinogens, since hepatomas have been pro- 
duced after nitrosamine. This is the first that 
hepatomas have been induced in monkeys and 
the first that a procedure has consistently pro- 
duced tumors in monkeys. (O'Gara, Kelly) 

2. The oncogenic response to carcinogens is 
increased in the lung if the pulmonary artery 
is ligated and an infarct produced in which the 
carcinogen appears to be trapped. (Stanton) 

3. Oncogenesis of the uterine cervix in 
hamsters produced by DMBA is inhibited if Vi- 
tamin A is given. (Chu) 

4. Special diets have a protective effect 
against carcinogens, but this can be overcome 
by larger doses of carcinogen. (Mulay) 

5. A strain of rats which carries a gene for 
congenital hyperbilirubinemia with jaundice is 



24 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



under study. A decrease in glucuronyl transfer- 
ase is found, and a similar condition is found in 
some human beings. An effect to inbreed these 
rats is making progress. A study on the effect 
of a chemical carcinogen, N-2-fluorylaceta- 
mide, is incomplete. Transplantation of a hepa- 
toma shows variation in the number of lung 
metastasis; none appear in the non-jaundiced 
rats, but they are numerous in the jaundiced. 
(Swarm) 

C. Biologic Factors of Neoplasia. — 1. Trans- 
plantation of a murine chondrosarcoma outside 
of the inbred strain of origin was successful in 
a well-differentiated tumor, and this may be 
due to the protective effect of the cartilage ma- 
trix. Cartilage survived after transplantation 
of a teratoid testicular tumor when other tis- 
sues were destroyed by the host, another indi- 
cation that the matrix protects from defense 
reactions of the host against foreign cells 
(Swarm) Clones of cells from the teratoid tu- 
mor obtained by tissue culture did not retain 
totipotency. (Sanford, Swarm) 

2. Metastasis of a mammary tumor was in- 
creased in C3H mice when orotic acid was giv- 
en. Thus a fundamental biologic factor in a 
neoplasm can be altered by a substance given 
to the host. (Chu) 

3. Irradiation of a chondrosarcoma produced 
the same effect if it was administered 24 hours 
before transplant, as when performed 24 hours 
after transfer. This offers a means for study- 
ing the primary effect of irradiation on a tu- 
mor and avoids the effect of irradiation on the 
host. (Swarm) 

D. Development of Spontaneous Tumors. — 
Studies on these tumors are important to deter- 
mine the natural incidence and pathogenesis. 

1. Spontaneous tumors are studied in vari- 
ous inbred strains of rats and in mastomys. 
The incidence of gastric cancer in mastomys 
appears to be genetic, and differs in 2 strains 
under study. (Snell) 

2. A number of transplanted tumors are 
maintained and are available to others. (Snell, 
Dunn, Swarm) 

3. An unusual contagious tumor in hamsters 
has been studied intensively in the past few 
years. This tumor is a reticulum cell sarcoma 
with leukemic manifestations, and can be 
transferred to other hamsters by feeding tu- 



mor tissue or cage contact. Recently the tumor 
was transferred by the bite of mosquitoes. It 
was proved that the mosquito transferred liv- 
ing cells and not a virus. This opens a new pos- 
sibility of cell transfer by insects, rather a vi- 
ral transfer. (Banfield) 

Viral Carcinogenesis 

Problems of immunology and resistance. The 
same interest in pathogenesis and the mecha- 
nisms of carcinogenesis will be found in the 
experiments carried out by pathologists with vi- 
ruses as was noted in experiments with chemi- 
cal carcinogens. Experiments now in progress 
are concerned with: A) Polyoma virus, B) 
SV40 and adenovirus 12, and C) Leukemogenic 
viruses. Other studies are also in progress on 
D) Immunology of virus tumors and E) Inter- 
feron. 

A. Polyoma Virus. — 1. Studies on the inter- 
action of mesenchymal and epithelial elements 
in organ culture, and the effect of the polyoma 
virus have continued. Major findings are that 
the submandibular gland whether rudimentary 
or adult will develop neoplasms if polyoma is 
added to the culture, but salivary gland mesen- 
chyme is necessary for the neoplastic change in 
the epithelial cells. Although hair-follicle tu- 
mors are found in mice with polyoma, hair-fol- 
licles to new syngeneic hosts remained in the 
growth phase of the cycle and did not develop 
tumors. (Dawe) Tooth buds infected by poly- 
oma and transferred to subcutaneous sites 
developed ameloblastomas. (Main, Dawe) 

2. After polyoma infection tumors found in 
the nasal fossa were proved to be from the neu- 
roepithelium. These tumors were similar to, 
but less invasive than the neuroepitheliomas 
induced in hamsters and rats by diethylnitrosa- 
mine (Herrold) and by 2,7-FAA (Snell) respec- 
tively. Since nasopharyngeal tumors are com- 
mon in parts of Indonesia, information on 
pathogenesis at this site is of interest. (Dawe) 

3. Polyoma virus has produced tumors in the 
brain of newborn hamsters. Relation between 
growth of virus and tumor induction has been 
determined. (Estable, Rabson) 

4. Hemangiomas induced in hamsters by 
polyoma are transplanted with difficulty in the 
first generation, but success increases in later 



r 



NATIONAL CANCER INSTITUTE 



25 



generations, and one line metastisized consis- 
tently. In early generations, the takes were less 
successful in polyoma infected hosts, but this 
resistance was overcome in later generations. 
Fish have been exposed to polyoma but no re- 
sults can be reported as yet. (Stanton) 

5. The behavior of a polyoma induced tumor 
in the hamster was compared with a sponta- 
neous and a carcinogen induced tumor after in- 
travenous injection of dissociated cells. The re- 
sulting nodules in the lung after injection of 
the polyoma tumor were interpreted as inflam- 
matory reactions rather than true neoplasms. 
(Herrold) 

6. Thymectomy was shown to increase the 
oncogenic effect of the polyoma virus. Of part 
ular interest was the observation that strains 
of mice resistant to polyoma oncogenesis were 
made susceptible by thymectomy and that sus- 
ceptible mice which do not respond to a strain 
of polyoma of low virulence did develop tumors 
when thymectomized. 

B. SVdj and adenovirus. — 1. Adenovirus 
growth in monkey kidney cells is enhanced by 
SV40. Other viruses tested failed to produce 
this enhancement. Adenovirus 12 induced tu- 
mors in C3H and BALB/c mice after thsonec- 
tomy on day of birth. Cells from rhesus mon- 
key were grown in tissue culture with Simian 
virus 40 and "transformation" noted, but cells 
failed to produce tumors when transferred 
back to the original donor. The possibility is 
suggested that the cells in culture had acquired 
an antigen from the virus, and that the monkey 
recognized this as foreign. These results indi- 
cate that infection with oncogenic viruses may 
be no risk in normal human beings, but a pos- 
sible hazard exists in persons with reduced im- 
munologic resistance as in thymectomized 
mice. (Rabson) 

2. Adenovirus 12 in green monkey kidney 
produces a characteristic nuclear stippling cells 
which is associated with active viral replica- 
tion only in the presence of SV40. Other virus 
combinations failed to produce this effect. (0'- 
Conor) The growth of both viruses in the same 
nucleus has suggested that "hybridization" and 
genetic recombination may occur. 
7C. Leukemogenic Viruses. — 1. Moloney virus 
infection in rats has disclosed previously 
inapparent Bartonella and encephalatozoon or- 



ganisms in the preleukemic state. (Stanton) 
Nucleic acids given preceding Moloney virus in- 
jection stimulated leukemogenesis. (Malm- 
gren) 

2. Rauscher virus has been given to mice in- 
fected with a malarial parasite that produces a 
severe anemia, and combined diseases will be 
studied. (Edcomb) Mice infected with the 
Rauscher virus survive longer than controls if 
given propylthiouracil. Thyroidectomy failed to 
produce this effect. (Dunn) A great prolonga- 
tion of life occurred in mice given blood trans- 
fusions after Rauscher virus. (Malmgren) 

D. Immunology and Resistance. — 1. Virus 
induced antigens are found in transformed 
cells from tissue culture although two strains 
of polyoma with different oncogenic potential 
were similar in their ability to produce anti- 
gen, yet the more oncogenic strain which was 
also thymotropic when injected into newborn 
animals inhibited the animal's ability to reject 
heterologous tumor transplants. This could 
have been because of injury to the thymus. 
(Friedman) 

2. A replicative form of an arborvirus has 
been found and characterized. It is now under 
study and should prove useful in determining 
the cellular location of RNA. (Friedman) 

3. Mechanisms of interaction of interferon 
and possible role in carcinogenesis are under 
study. The action appears to involve active pro- 
tein synthesis. Interferon appears to limit pro- 
duction of viral RNA. (Friedman) 

Accumulation of Data (Laboratory 
Animals and Other Species) 

Because the training of pathologists is not 
restricted to one organ system or type of dis- 
ease, they are often able to contribute to the 
general knowledge of the normal and patho 
logic anatomy of many different animal species. 
This contribution is often incidental to the 
main purpose of cancer research, yet it is indis- 
pensable to the intelligent use of biologic ma- 
terial. Unless the normal anatomy and sponta- 
neous pathologic alterations are recognized, it 
is impossible to interpret the effects of experi- 
mental procedures. 

A. Information on Laboratory Animals. — 
1. Information is continually being added on 
inbred strains of rats, and more recently on 



26 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



mastomys. In this species the female has a well 
developed prostate gland ,which will be des- 
cribed in a forthcoming publication. (Snell) 

2. A guide to the normal and pathologic anat- 
omy of the laboratory mouse is being prepared. 
(Dunn) 

3. Complete autopsies are being performed 
on both control and experimental monkeys and 
much needed information is being gathered 
about these primates. A form of vascular dis- 
ease resulting from subcutaneous injections of 
polycyclic hydrocarbons has been described. 
(O'Gara) 

4. Collagenous connective tissue and elastin 
from the embryo to old age in man and labora- 
tory animals is being studied. A new staining 
and extracting procedure for elastic fibers us- 
ing KMn04 is being used in this study. (Ban- 
field) 

5. The maintenance of fish within the labo- 
ratory and use for experimental carcinogenesis 
has been accomplished. (Stanton) 

6. The normal estrous cycle has been estab- 
lished by vaginal smears for Sprague-Dawley 
and Fischer rats, C3H mice, Swiss, and DBA 
mice, hamsters and guinea pigs, and the effect 
of constant light and darkness on the cycle in 
some of these animals has been determined. 
(Chu) 

B. Phylogenetic Aspects of Neoplasia. — 1. An 
important project has been started on phy- 
logenetic aspects of neoplasia in cooperation 
with the Smithsonian Institute and Marine Bi- 
ology. The collection and identification of neo- 
plasms in invertebrates has been started, a lit- 
erature survey now having over 300 entries 
will be continued and a registry of specimens 
will be made. Neoplasia in planaria and cock- 
roaches is being investigated under laboratory 
conditions. It is already apparent that adequate 
criteria have not been used previously to deter- 
mine neoplasia in lower forms. Tumors have 
been found in fish from a lake where no out- 
board motors were used, thus eliminating mo- 
tor oil as the only carcinogen in fresh-water 
lakes. (Dawe) 

Collaborative Research 

It is recognized that many research projects 
at the National Cancer Institute require the col- 



laboration of a pathologist, especially in the 
final evaluation of the effect of an experimental 
procedure on laboratory animals. The Labora- 
tory of Pathology has always tried to make this 
assistance available. 

A. Methods 1. The pathologist may take 
an active part in planning an experiment and 
in following it through; he may take the re- 
sponsibility for all autopsies and histologic 
diagnoses in an experiment; he may review 
only the histologic sections in a given experi- 
ment; or he may serve as a consultant to re- 
view selected material with no responsibility 
for the entire experiment or its publication. 
Finally, he may make use of material accumu- 
lated by other investigators for independent 
studies concerning pathologic alterations. It is 
emphasized that full collaboration of the pathol- 
ogist at the time the experiment is planned is 
the most satisfactory arrangement for it in- 
sures the best and most economical selection of 
material for pathologic studies. 

2. In addition to the use of the light micro- 
scope and standard autopsy procedure, individ- 
ual members of the Laboratory of Pathology 
have become proficient in special techniques 
such as fluorescent antibody visualization, elec- 
tron microscopy, tissue culture, autoradi- 
ography, exfoliative cytology, special cytology, 
and histochemistry. These special skills are of- 
ten made available in collaborative studies. 

B. Examples. It would be tedious to con- 
sider all the collaborative work now in prog- 
ress in the Laboratory of Pathology, and this 
should be unnecessary since it is covered in 
reports from other laboratories. However the 
following are noteworthy : 

1. Fluorescent antibody studies by Dr. Malm- 
gren. He has studied a) plasma cell neoplasms 
and other conditions in man and animals in 
collaboration with Dr. Fahey ; Dr. Solomon and 
Dr. Brecher; b) localization of antibodies in 
mycosis fungoides with Dr. Kenneth Blaylock; 
c) Antigens in human leukemic cells (with Dr. 
Mary Fink, Frank Rauscher, Henry Orr, and 
Myron Karon). 

2. Dr. Dunn has continued collaborative stud- 
ies with Dr. Andervont on mammary tumors; 
with Dr. Evans on the effect of transfer of tis- 
sue cultures to the anterior chamber of the eye; 



NATIONAL CANCER INSTITUTE 



27 



with Dr. Law on thymectomized and leukemic 
mice; and has diagnosed sections for many- 
other investigators both in and out of the NCI. 

3. Dr. Banfield has used the electron micro- 
scope to assist Dr. Hendler, Dr. Tani and Dr. 
Kuff with identification of cellular fractions; 
with instrument engineering to develop a tis- 
sue changer for E.M. ; with Dr. McMaster to de- 
termine if antibodies against tobacco mosaic 
virus occur in man; with Dr. Steinberg and 
Challoner to correlate morphologic changes 
with the biochemical effects of a high fatty acid 
concentration and with Dr. Laster, Nadel and 
Reuber in E.M. investigation of normal and 
pathologic tissues. Findings are that macro- 
phages persist in Whipple's disease; cytoplas- 
mic crystals were found in presumed leukemic 
cell of guinea pig, but no virus ; Fabry's disease 
was diagnosed by E.M., but the diagnosis was 
missed with light microscope. 

4. Dr. Snell is frequently consulted regard- 
ing the normal and pathologic anatomy and 
neoplasms in laboratory rats. 

5. Dr. Stewart frequently gives an opinion 
regarding lesions observed by other investiga- 
tors at the NCI, and from outside. 

6. Dr. O'Gara is becoming recognized as an 
authority on the pathologic anatomy of the 
monkey, and assists others in this field. 

7. Dr. Reuber has collaborated with Dr. Har- 
old Morris on studies of minimal deviation tu- 
mors. 

LABORATORY OF PHYSIOLOGY 

Office of the Chief 

The mechanism of carcinogenesis by ultra- 
violet light is being pursued by Dr. Blum. He 
has been concerned with obtaining and analyz- 
ing data from experiments on epidermal hyper- 
plasia of mouse skin induced by ultraviolet 
light. In counting numbers of epidermal cells 
and of mitotic figures, a good deal of variabil- 
ity is found, and in spite of the application of 
a method of random sampling, subjective judg- 
ment of the observer cannot be eliminated. 
Conclusions therefore must be tentative but the 
results suggest that the effects of ultraviolet 
light are mediated by some substance diffusing 
beyond the limits of the immediate photochemi- 



cal effects of the radiation. Studies of carcino- 
genesis in albino mice with ultraviolet wave- 
lengths that penetrate little below the epi- 
dermis have resulted in the induction of 
epidermal tumors only. This agrees with Dr. 
Blum's earlier studies to indicate that the carci- 
nogenic effect of ultraviolet light is confined to 
the immediate site of action of the radiation, in 
apparent contrast to the case of transient hyper- 
plasia. Interest has been maintained in the haz- 
ards of cancer of human skin from ultraviolet 
light, particularly that of sunlight, and the 
bearing of these experimental studies there- 
upon. 

New experiments by Dr. White and his asso- 
ciates to determine the effect of tumor protein 
as a source of dietary nitrogen on the "protein 
reserves" of normal and tumor-bearing rats 
are now under investigation. Sprague-Dawley 
rats ingesting a low casein (6%) diet develop 
severe cirrhosis of the liver between 87-150 
days of feeding. When fed 8% casein diet, cir- 
rhosis was less severe. Even after 145 days 
many of the animals showed only moderate 
cirrhosis. Osborne-Mendel rats on 6% casein 
diet showed a much less severe cirrhosis up to 
262 days; but on an 8% diet no cirrhosis was 
observed up to 210 days. At the end of 365 
days, 15 of the 22 animals had cirrhotic lesions. 
Animals showing moderate to severe cirrhosis 
showed no reduction of these lesions when 
transferred to an 18% casein diet, indicating 
the damage was not reversible. Studies using 
tumor protein as a source of nitrogen are being 
compared with the above results. Attempts will 
be made to determine whether the cirrhotic 
effect can be reversed by other agents such as 
choline. 

The office of this Laboratory has performed 
in a superior manner. In addition to the smooth 
administrative operation of the Laboratory, 
they have met deadlines, crash programs, last- 
minute manuscripts and the like, with equally 
superior fashion. All members have willingly 
worked long hours, without request for over- 
time or compensatory leave. Financial records 
have been kept on a weekly basis, making avail- 
able to the Chief current expenditures and those 
to be encumbered. 

The machine, electronics and glass shops of 
this Laboratory have made endless experiments 



28 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



possible by design, engineering and fabrica- 
tion. They have modified and repaired existing 
equipment which has saved many experiments 
and much valuable time. These shops have been 
available to other laboratories in the NCI and 
here too they have rendered valuable assist- 
ance. 

It is indeed heart-warming to know that the 
administrative and shop personnel of this Lab- 
oratory have responded so enthusiastically and 
efficiently with their services. 

Cancer Physiology Section 

Dr. S. H. Wollman and research associates 
Drs. G. Andros and J. E. Loewenstein continue 
to study the mechanism of the iodide concen- 
trating mechanism of the thyroid gland. They 
have been exploring some aspects of intrathy- 
roidal heterogeneity which may help explain 
kinetics of radioiodine metabolism in the whole 
gland. Studies involving a peculiar type of cys- 
tic follicle in the thyroid of the C3H mouse 
showed that although it did not accumulate or- 
ganic iodine it could concentrate inorganic ra- 
dioiodide to about the same extent as nonnal 
follicles. This cystic follicle also was responsive 
to TSH (thyroid stimulating hormone) stimu- 
lation in ways similar to the normal follicle. 
They have also studied intrafollicular hetero- 
geneity, especially of the colloid, and have ex- 
plored the eifect of factors such as dietary 
iodine intake which influence the rate of diffu- 
sion of iodoproteins in the lumen of the follicle. 

Dr. Rabinovitz and research associates Drs. 
Honig and Waxman are studying in both nor- 
mal and tumor cells the path that amino acids 
follow leading to the synthesis of proteins and 
the controls exercised by the cells in regulating 
these processes. With Dr. Honig, Dr. Rabino- 
vitz has been studying the inhibition of protein 
synthesis in Sarcoma 37 ascites cells by actino- 
mycin D. Incubation of S37 cells in the pres- 
ence of antinomycin D using pyruvate as a 
source of oxidizable substrate but lacking in 
glucose, leads to a marked inhibition of protein 
synthesis after 90 minutes. Cytoplasmic nu- 
clearproteins were equally inhibited. This inhi- 
bition could be prevented and relieved by the 
addition of glucose. These data indicate that 
actinomycin D is not acting by reducing the 



availability of messenger RNA directly re- 
quired for protein synthesis, but is probably in- 
terfering with some ribonucleic acid required 
for utilization of amino acids under respira- 
tory conditions. Similar observations were 
made with human chronic myelogenous leuke- 
mic cells. With Dr. Waxman he has been study- 
ing the role of iron and heme in maintenance 
of polyribosome structure and regulation of 
globin synthesis of rabbit reticulocytes. It is 
now generally accepted that the ribosomal ag- 
gregates (polyribosomes) are the sites of glo- 
bin synthesis in reticulocytes as well as of a 
variety of proteins in other cell types. They 
have demonstrated that polyribosomes of retic- 
ulocyth-s can be disaggregated under condi- 
tions which bring about iron depletion, such as 
preincubation with amino acids to remove avail- 
able iron by hemoglobin synthesis by binding 
the iron in cells with chelating agents. Poly- 
ribosomes do not disaggregate in cells when 
incubated with low concentrations of ferrous 
iron or hemin and under appropriate condi- 
tions disaggregated polyribosomes can be reas- 
sociated from ribosomes. Optimal hemoglobin 
synthesis is only seen in cells having high poly- 
ribosome levels and it is through the mainte- 
nance of the active aggregated state of ribo- 
somes that heme supports the synthesis of 
globin. The mechanism at the molecular level for 
this cytoplasmic regulation of protein synthesis 
by a nonprotein component, heme, is not pres- 
ently understood nor is it readily explained by 
the currently accepted theory of the mecha- 
nism of polyribosome action. 

Dr. Reid continues his studies on the urinary 
excretion patterns in leukemia. This study re- 
quires the employment of chemical and mathe- 
matical techniques. The chemical procedures 
comprise an integrated chromatographic sys- 
tem developed by Dr. Reid which separates a 
urinary specimen into a large group of mixed 
fractions. The mathematical techniques which 
are primarily those of linear algebra represent 
an approach to further resolution of these mixed 
fractions by means of computational anal- 
ysis of their ultraviolet absorption spectrum. 
The large volume of data thus generated is 
handled by a data processing machine. This ap- 
proach to studying purine and pyrimidine ex- 
cretion products in leukemia is extremely time 



NATIONAL CANCER INSTITUTE 



29 



consuming and fraught with ever-rising prob- 
lems. Although publications from this type of 
approach are not frequent, it requires one with 
the patience, knowledge of chemical, mathemat- 
ical, and electronic procedures which Dr. Reid 
possesses to bring it to a successful conclusion. 
Several urinary components have been isolated 
which appear to be identical with pseudouri- 
dine itself except it gives no orcinol response 
and on electrophoresis and thin layer chroma- 
tography exhibits evidence of complex forma- 
tion. It is suggestive of some isomer of pseu- 
douridine. Work is continuing on the urinary 
profiles of different leukemic subjects but ulti- 
mate identification of peaks is needed before 
patterns can be established. 

Energy Metabolism Section 

The investigations on the total energy expend- 
iture of the tumor-bearing rat as it is derived 
from both the host and the tumor have been 
continued by Drs. Morrison, Millar and Pratt. 
Present investigations are concerned with the 
patterns of heat production with concomitant 
recording of feeding behavior and other activ- 
itj?^ to better understand the mode and extent 
to which feeding behavior influences heat pro- 
duction and the impact that the presence of a 
tumor in various stages of growth has on the 
energy exchange. Recent observations indicate 
that in the normal rat there appears to be no 
distinction in terms of energy expenditure or 
activity pattern between the energy expendi- 
ture resulting from feeding activity and that 
resulting from non-feeding activity. Thus it ap- 
pears likely that all non-resting energy expendi- 
ture will be equally affected by the potential of 
dissipating heat. 

The energy metabolism pattern of normal rats 
is now being studied at various environmental 
temperatures. These experiments will deter- 
mine whether the partition of total expenditure 
into rest, feeding activity and non-feeding activ- 
ity is also altered. This may be relevant to the 
depression of food intake of the tumor-bearing 
animal which partially recovers its food intake 
on exposure to low environmental tempera- 
tures. Experiments are in progress on the meta- 
bolic pattern of rats after destructive lesions 
of the lateral hypothalamus, particularly on the 
relative changes in water electrolyte exchange 



and nitrogen in tissue experiments both in the 
normal and tumor-bearing animals. Experi- 
ments on the etiology of the development of 
gastric ulcers in the rat during tumor growth 
have thus far indicated that the incidence of 
glandular ulcers in the presence of a transplant- 
ed tumor is a function of the condition of the car- 
cass as influenced by a combination of circum- 
stances (diet, tumor size and time) whereas 
ulcers of the forestomach are possibly related 
to stomach contents. 

Dr. Pratt and Mr. William White have made 
rapid and important progress in developing 
mathematical and computer programming 
techniques which may be applicable to the in- 
vestigator in the biomedical field involving in- 
formation storage and retrieval problems. In 
collaboration with Dr. L. Thomas of the Labo- 
ratory of Pathology, a complete computer- 
based system has been devised for storing and 
retrieving of surgical pathology, autopsy pa- 
thologjr and cytology records. This affords the 
staff a rapid and precise identification of study 
material and provides a complete description of 
disease pathology. Similar programming sys- 
tems have been devised for NCI and NIH 
grants. The former is in progress and formula- 
tion and computer programming of such 
grants should soon be completed. The latter 
consisted of a complete system analysis of the 
Division of Research Grants relative to the stor- 
age and retrieval of scientific content of the 
NIH Research Grants. This vast undertaking 
was completed in a relatively short period of 
time. Dr. Pratt in conjunction with Mrs. J. N. 
Toal has continued their mathematical and com- 
putational dealing with the ultraviolet spec- 
trometric analysis of oligonucleotides. Previous 
reports of this successful approach for the anal- 
ysis of the base composition of oligoribo- 
nucleotides have resulted in new experiments to 
apply this technique to determine nucleotide se- 
quence simultaneously. Insufficient work has 
been done to determine if the computational 
approach will be a useful adjunct, laboratory 
procedure. 

Physical Biology Section 

The characterization and comparison of the 
nucleic acids and nucleoproteins of normal and 
malignant tissue are under investigation by Dr. 



30 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



Shack. A good part of the time has been spent 
on methodology involving centrifugal and elec- 
trophoretic procedures so that quantitative iso- 
lation and subsequent fractionation could be 
carried out with microgram rather than milli- 
gram amounts of DNA. This has been achieved 
through the isotopic labeling of DNA. It has 
now been possible to separate a trace of double 
stranded DNA from a large amount of single 
stranded DNA and a trace of single stranded 
DNA from a large excess of double stranded 
DNA. This procedure of separation was of par- 
ticular significance in the attempt to separate 
and concentrate certain species of DNA such as 
viral DNA. Work is in progress to attempt to 
determine whether DNA of viral origin is pres- 
ent in tumors caused by viruses. 

Dr. Breitman is continuing his work on the 
control of ribo- and deoxyribonucleotide syn- 
thesis. Studies on the metabolism of two thy- 
mineless strains derived from E. coli have con- 
tinued. He plans to develop an assay for ribonu- 
cleotide reductase activity, the enzyme system 
which synthesizes deoxynucleotides from ribo- 
nucleotide precursors; to elucidate the mech- 
anism of thymine to thymidine conversion; 
and to establish the exact action of thymidine 
on ribonucleotide synthesis. 

Radiation Biology Section 

Studies on the repair of sublethal damage in 
X-irradiated mammalian cells by Dr. Elkind 
have been extended. He has shown: (a) That 
the repair of damage is weakly dependent on 
temperature; some repair is evident even at 
1°C. (b) Repair of damage is not an oxidative 
process; repair proceeds quite well under the 
simultaneous conditions of hypoxia and subop- 
timal growth temperatures. And (c), the gross 
effects of irradiation on macromolecular syn- 
thesis show that the pattern of DNA synthesis 
is strongly affected. The patterns of RNA and 
protein synthesis are weakly affected. In con- 
trast, the RNA inhibitor, actinomycin D, has a 
strong effect on the repair process while DNA 
(5-fliuorodeoxyuridine) and protein (puromy- 
cin) inhibitors have minor effects. 

The quantitative studies of radiation damage, 
spontaneous recovery and induced recovery 
on the hemopoietic system are actively being 
pursued by Dr. W. W. Smith. More recently 



she has found that if endotoxin is given before 
or shortly after irradiation, the number of col- 
ony-forming units arising increases endogen- 
ously in the spleen. Endotoxin given to nonirra- 
diated animals increases the number of colony- 
forming units in the spleen (determined by 
injection into irradiated recipients) . Endotoxin 
causes a relatively early return to normal in 
both spleen and bone marrow forming units. 
Experiments are underway to determine 
whether or not the numbers drop as low in 
treated as in untreated animals. Kinetics of the 
recovery process in animals treated after irra- 
diation are underway. 

Dr. Draper is pursuing his studies on anti- 
body formation following multiple irradiation 
and immunization procedures. The goal of this 
research is to determine the sources of natural 
and specific hemolysin during the recovery 
from irradiation damage and whether there is 
a selection site of production of antibody type 
following irradiation exposure. A modification 
of a method for the determination of the avid- 
ity of hemolysis produced in rabbits has been 
made which permits the use of the same prepa- 
ration of untreated cells for both cell popula- 
tions used in the system and thereby eliminates 
the possibility of differences between them 
with respect to their affinity for the hemolysin 
or their susceptibility to spontaneous or im- 
mune lysis. The relevance of this parameter, 
that is hemolysin transfer to avidity, has been 
verified by dilution effect experiments in which 
the avidity of antibody is measured in terms of 
the persistence of antigen-antibody complexes 
upon the dilution of the reaction mixture. The 
less avid the antibody, the higher its rate of 
dissociation from antigen: dilution reduces the 
probability of reassociation. Thus the reduction 
in the number or proportion of complexes is a 
function of the dissociation rate of the anti- 
body. The dilution effect is minimal with avid 
hemolytic antibody showing little or no ten- 
dency to transfer and vice versa. 

Dr. Riesz has recently been concerned with 
the effects of ionizing radiation on biologically 
active macromolecules. In collaboration with 
Dr. F. H. White, NHI, they have been determin- 
ing the eflfect of radiolysis on dry ribonuclease. 
They have devised a new method for studying 
the distribution of free radicals in irradiated 



NATIONAL CANCER INSTITUTE 



31 



macromolecules in vacuo. Stable free radicals 
are produced and reacted with tritiated hydro- 
gen sulfide. Employing electron spin resonance 
measurements, about two-thirds of the carbon 
radicals are removed in less than 20 minutes at 
room temperature and the remainder can be 
removed by heating to 70° C. Thus the distribu- 
tion of carbon-bound tritium among the amino 
acid residues is presumably a good approxima- 
tion of the original radical distribution pro- 
duced as indicated above. Following the remov- 
al of exchangeable tritium bound to oxygen, 
nitrogen and sulfur, the distribution of carbon- 
bound tritium among the amino acid residues 
can be determined. The carbon-bound tritium 
incorporated into amino acids does not corre- 
late with the number of primary, secondary or 
tertiary carbon-hydrogen bonds in a given 
amino acid residue and free radical formation 
proceeds in a non-random fashion. It seems 
likely therefore that the distribution of free 
radicals depends on the conformation of the 
polypeptide chain. 

Dr. Charles R. Maxwell has undertaken with 
Dr. Elizabeth S. Maxwell, NIAMD, a study of 
the effect of ionizing radiation on the cell to 
determine at what site the damage occurs and 
its effect on normal function. These studies will 
entail work on the ribosome, transfer ENA and 
protein synthesis. This initial work will be 
done on the rat liver homogenate. 

LABORATORY OF CHEMICAL PHARMA- 
COLOGY 

The research work in the Laboratory of Chem 
ical Pharmacology continues to stress physio- 
logical disposition and distribution of anti- 
neoplastic agents. 

Folic acid antagonists represent one impor- 
tant class of chemotherapeutic compounds. A 
number of pteridine precursors have been suc- 
cessfully synthesized and 3'-iodoaminopterin 
has been prepared. This compound, while not 
importantly active in L1210, could be of use in 
tracer studies if an iodine radioactive isotope 
can be substituted. The studies of the physio- 
logical disposition of methotrexate in animals 
and man reported last year have been contin- 
ued. More animals and more patients have been 
added. No unexpected results have been ob- 
tained. The observation that oral administra- 



tion of large doses of methotrexate results in 
slow and incomplete absorption, has been 
amply confirmed. This pharmacological obser- 
vation is important in view of the observation 
by the Leukemia B Group that intermittent 
oral methotrexate may be as effective as inter- 
mittent parentei'al methotrexate in childhood 
leukemia. Perhaps the long continued absorp- 
tion of a smaller amount of methotrexate is as 
toxic and as therapeutically active as the rapid 
absorption of a larger amount of methotrexate. 
It is interesting to note that in plasma levels in 
man after 100-fold, different i.v. doses are par- 
allel and have the same volume distribution. 
Studies on the plasma protein binding of metho- 
trexate have been performed. Approximately 
50% of the plasma concentration of metho- 
trexate is bound to the plasma proteins, largely 
albumin, in mouse, rat, dog, and man. Of par- 
ticular interest is that a number of weak organ 
acids, including the salicylates, sulfonamides, 
and probenecid, can displace methotrexate from 
plasma protein. This can result in increased 
lethal toxicity when a long acting sulfonamide, 
sulfamethoxypyrimidine, and methotrexate 
are administered simultaneously. Studies 
of the displacement of dichloromethotrex- 
ate have not yet been performed. Since this 
agent is 90-95% bound to plasma protein, dis- 
placement could have serious toxicological 
effects. In fact, the possibility exists that some 
of the erratic toxicity reported after dichloro- 
methotrexate administration in man might be 
a result of concomitant administration of aspi- 
rin, sulfonamides, penicillin, and so forth. 
These studies will be followed with great inter- 
est. Basic physico-chemical studies of the rela- 
tionship to protein binding and the solubility of 
a variety of relatively insoluble organic amines 
has been performed. It is interesting to note 
that the protein binding and aqueous solubility 
of these compounds correlates well. This sug- 
gests that the solvation of the protein and the 
chemical might be related to the mechanism of 
reversible protein binding. 

The use of methotrexate in ventriculocister- 
nal or ventriculoventricular perfusions for 
meningeal leukemia or intracranial neoplasms 
has been extended this year. Nine patients 
have been studied with 30 or more perfusions. 
A maximum dose of 75 micrograms per milli- 



32 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



liter of methotrexate in 500-1000 milliliters 
has been used. No overt toxicity has been seen 
and, in the patients who have come to autopsy, 
no histologic evidence of toxic effects of metho- 
trexate have been seen. The solid tumors have 
not been of such a type that therapeutic evalua- 
tion has been possible. However, in the two 
patients with meningeal leukemia, complete 
remission of the meningeal leukemia was ob- 
tained. In one patient, who died some months 
after perfusion, no leukemic cells were found 
at autopsy in the brain. 

In an interesting study total body pyrimi- 
dine utilization was studied in chronic leukemic 
patients given 6-azauridine. The surprising 
finding was that 6-azauridine increased pyrmi- 
dine utilization during treatment. Perhaps this 
is a reflection of the inactivity of 6-azauridine 
against human neoplastic processes. 

The metabolism of hydroxyurea has been ex- 
tensively studied. A significant proportion of 
the hydroxyurea is converted to urea but inter- 
estingly enough 10% is excreted as carbon 
dioxide. The mechanism of transformation of 
hydroxyurea to carbon dioxide is not as yet un- 
derstood. 

Preliminary studies have been performed 
with cj^osine arabinoside. The one significant 
finding has been the demonstration that an ab- 
normal cytosine nucleoside was a contaminant 
of a commercially produced cytidylic acid. This 
developed out of a study of cytidylic acid con- 
cerning its ability to reverse cjdosine arabino- 
side's antitumor activity. This "cytidylic acid" 
was found to have antitumor activity itself. 
The toxicity of cytosine arabinoside in dogs 
and monkeys is consistent with the human ob- 
servations that the major effects are on the 
granulocytes. Otherwise the toxicity was not 
remarkable. 

Methyl isopropylcarbamyl benzyl hydrazine 
(MIH) has been studied with respect to its 
biochemical degradation in the body and its 
distribution. The degradative pathway seems 
clearly to involve an azo compound and methyl- 
hydrazine. Considerable amounts of p-isopropyl- 
terephthaic acid are excreted as well as a 
variety of other small molecular weight related 
compounds. Certain intermediates may inhibit 
monamine oxidase. Studies on the carcinogenic- 
ity of MIH have been shown to occur in a 



number of species of mice. C57 black strains 
are relatively resistant. In rats, MIH causes 
mammary tumors as well as a great variety of 
adenomas and carcinomas. The variety of tu- 
mors seen in some of the rats given MIH is 
reminiscent of the tumors seen in hamsters giv- 
en polj^oma virus. Studies of the carcinogenesis 
of MIH in the rhesus monkey are under way. 

Two methods to determine the physiological 
disposition of bis-j8-chloroethylnitrosourea 
(BCNU) have been employed this year. One 
method, a chemical method, has shown that the 
unaltered molecule rapidly entered the cerebro- 
spinal fluid. It disappeared from blood and 
spinal fluid quite rapidly, and only traces were 
found in the urine. Studies using radiolabelled 
BCNU (C" in all four chloroethyl carbons) 
have shown that in the mouse, while most ra- 
dioactivity appeared in the urine, a significant 
amount was in respiratory CO2 and small but 
significant amounts remained in liver and kid- 
ney. In the monkey, considerable amounts of 
radioactivity remained in various body tissues 
after 24 hours. It is notable that measurable 
plasma levels of radioactivity were found as 
long as 72 hours after a single dose of BCNU. 
It is clear, of course, that this does not persist 
as unaltered BCNU. It will be interesting to 
determine what these degradation products 
are. The ultrastructure changes following 
BCNU in liver, kidney, lung, and intestine of 
rats treated with this agent have been investi- 
gated. Perhaps the most significant observation 
which might relate to the delayed toxicity of 
BCNU is the profound effect seen on the blood 
vessels of the involved organs. This is manifest 
by a thickening of the walls and obliteration of 
the small vessels. Other important effects seen 
were desquamation of the renal tubules, unex- 
plained dense inclusion bodies in the cytoplasm 
of hepatic cells and intestinal mucosa cells. A 
variety of other nitrosoureas have been stud- 
ied. The other agents so far studied are 
fraught with problems related to their physical 
characteristics such as low water solubility and 
instability. Thus far no agent seems superior 
to BCNU. One interesting observation is that 
BCNU in cottonseed oil appears to be stable 
for long periods of time. The possibility exists 
that this might be a useful means of adminis- 
tering intramuscular BCNU. 



NATIONAL CANCER INSTITUTE 



33 



We also studied some of the properties of di- 
methylsulfoxide (DMSO) this year, although 
these studies were not reported in Life, Time, 
or Newsweek. DMSO is an agent which has 
had many spectacular attributes claimed, in- 
cluding a variety of pharmacological actions in 
addition to that of being an excellent solvent. 
One of the claims was that it enhanced the per- 
meation of compounds through cellular mem- 
branes. If this could be substantiated, it would 
be a great help in the problem of attacking the 
pharmacological hideouts of tumor cells. We 
studied the ability of DMSO to enhance the cel- 
lular penetration of a variety of pharmacologic 
agents. These included the entry of para-amino- 
hippurate into the spinal fluid and in brain as 
well as into other tissues and the oral absorp- 
tion of morphine, insulin, penicillin, and a 
number of other drugs. These studies were uni- 
formly negative. No difference in concentration 
ratios or in intensity or duration of pharmaco- 
logical action could be found when the com- 
pound was dissolved in DMSO as opposed to 
solution in water alone. Interest in this area 
leads us to study tetramethylurea, another 
agent which might be a useful solvent. Tetra- 
methylurea does have excellent solvent proper- 
ties. However, its toxicity, though not great, is 
significantly greater than DMSO. Interestingly 
it has a small amount of anti-tumor activity in 
certain experimental mouse tumors. 

Studies on the mechanism of action of nitro- 
gen mustard on DNA have been continued. The 
finding reported last year that the sensitive E. 
coli cells, i.e. sensitive to nitrogen mustard, ex- 
hibit a lack of ability to repair the crosslinks 
caused by the nitrogen mustard. This year's 
studies have been directed more towards the 
kinetic of reaction between nitrogen mustard 
and double stranded DNA. These kinetics seem 
to be essentially first order after a pronounced 
lag. These observations are explained by the 
notion that one crosslink will denature DNA. 
The lag is a function of the time it takes to 
make the first reaction between the chloroethyl 
group and the base and the first order kinetics 
then follows as the second joins the opposite 
base. These studies are interesting in their use 
of a variety of sophisticated techniques includ- 
ing the IBM 620 computer to validate the 
mathematical models. 



Of increasing importance, it seems to us, will 
be the use of human and animal cell lines 
grown m vitro. By the use of a combination of 
DMSO and DNAase, freezing and recovery of 
the Grace human myelogenous leukemia cell 
line and a variety of other cell lines has been 
shown to be feasible. If attention is paid to the 
details, this is a practical and simple method. 
An important development has been the perfec- 
tion of a method of synchronization of mitotic 
events in tissue culture. This method utilizes 
the changing adhesiveness of cells during the 
mitotic cycle to extract a population of cells ho- 
mogeneous with respect to their mitotic cycles. 

The development of hepatic carcinomas in 
five monl^eys given dimethylnitrosamine has 
not only been of interest in terms of questions 
of susceptibility of primates to carcinogenesis, 
but provides a new experimental tool in which 
studies on hepatomas may be performed. In 
collaboi'ation with the Surgery Branch and the 
Diagnostic Radiation Branch, angiograms have 
been used to demonstrate the extent of the 
hepatomas in certain of these animals. In addi- 
tion the possibilities of direct cannulation of 
hepatic vessels and the treatment of these 
hepatomas with a variety of appropriate anti- 
neoplastic agents is being actively pursued. 

Combination toxicitj' studies of anticancer 
agents are well under way. No specific infor- 
mation of any startling variety is yet available. 
The difficulties of designing and interpreting 
these experiments are slowly being overcome. 
The importance of combining agents with inde- 
pendent action is becoming more apparent. An 
urgent need is seen to adapt the Skipper cell 
kill model to the P1798 tumor which is suscep- 
tible to all of the clinically useful leukemic 
agents and then to use this system in combina- 
tion experiments to allow for the estimation of 
therapeutic effectiveness and host toxicity in 
parallel closely related experiments. 

Studies designed to elicit distribution of anti- 
cancer drugs in a variety of potential phar- 
macological hideouts such as the thymus gland, 
the center of necrotic tumors, and so forth 
awaits the installation of the ultra-microtone 
which will allow whole animal autoradiography 
of medium sized rats. 

An important activity of the chief of this la- 
boratory (with substantial assistance from a 



34 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



variety of staff members) has been participa- 
tion in a committee organized by the Director 
of the National Institutes of Health to consider 
overall problems of the state of the art of the 
pharmacology today and to determine whether 
strenuous efforts should be made by the NIH to 
enhance the effectiveness of this important dis- 
cipline. This has resulted in a scientific paper 
embodying certain important recommendations 
which will appear in Science in the near fu- 
ture. Out of this, I think, has come an aware- 
ness that the important concepts in pharmacol- 
ogy are related to the interaction between chem- 
ical substances and biological material and 
have many implications in most aspects of bio- 
medical research. Implicit in this is the aware- 
ness that one must look beyond the intimate 
effect of a chemical agent on some specific and 
discrete biological or biochemical system and 
be aware of potential implications for not only 
the organ system but the whole animal, clinical 
therapeutics, medical practice, and public 
health in general. 

MEDICINE BRANCH 

The 1964-65 fiscal year has been a very 
eventful one for the Medicine Branch of the 
NCI in many respects. A major administrative 
change occurred when Dr. Emil Frei resigned 
to accept an important position at the Univer- 
sity of Texas M.D. Anderson Hospital in 
Houston, Texas. Dr. Paul Carbone was ap- 
pointed the Head of the Solid Tumor Service, a 
service which was created last year when the 
clinical program was divided into 2 sections. 
The other section, the Leukemia Service, con- 
tinues under the direction of Dr. Emil Frei- 
reich. 

One of our senior investigators. Dr. Robert 
H. Levin was killed in an airplane accident. He 
was a young and bright investigator who had 
already made a number of important contribu- 
tions in the clinical study of acute leukemia. 

Excellent and close collaboration has been 
maintained between the Laboratory of Chemi- 
cal Pharmacology and the Medicine Branch. 
This is one of the most important and mutually 
beneficial interactions in which the Medicine 
Branch participates. 

In the past 12 months a number of problems, 
largely administrative, have arisen with the 



Chemotherapy Tumor Group of the Baltimore 
U.S.P.H.S. Hospital which has prevented an 
effective collaborative effort. Without the avail- 
ability of this facility it has not been possible 
for the Medicine Branch to perform the 
number of Phase I and II chemotherapy stud- 
ies which are necessary in a major clinical anti- 
tumor therapy program. Hopefully these prob- 
lems will be resolved in the near future. 

Activities in the Solid Tumor Service have 
gradually become disease-oriented during the 
past year with emphasis on the Ijonphomas 
particularly Hodgkin's Disease and on the tes- 
ticular tumors. This is in addition to the inter- 
est in intensive chemotherapy of the blastic 
crisis in chronic myelocytic leukemia. 

Lymphomas 

Several important findings have emerged in 
patients with lymphomas including Hodgkin's 
Disease. An intensive combined drug and ra- 
diation study in 14 patients with Hodgkin's Dis- 
ease was completed. They were treated with a 
combination of vincristine, cyclophosphamide, 
methotrexate, and prednisone given in two 
week courses and combined with radiotherapy 
in patients with Stage I or II disease. Moderate 
reversible toxicity occurred in all patients but 
tumor regression was attained in 13 of the 14 
patients with chemotherapy alone prior to ra- 
diotherapy. Treatment was not successful in 4 
of the patients — all in Stage III (out of 10 in 
that category). Most of the patients are still 
being followed but a significant finding to date 
is that in the responsive patients with Stage 
III Hodgkin's Disease the median duration of 
unmaintained remission is approaching 10 
months. In the other stages, the median of un- 
maintained remission is over 12 months. This 
is considerably better than with any other 
known drug. This study is continuing with a 
slightly different combination of drugs but 
with the same objectives in mind. In the new 
study patients with the other lymphomas are 
also included. 

A large cooperative study of lymphomas 
with the Eastern Solid Tumor Group and the 
Acute Leukemia B Group is being conducted 
under the co-chairmanship of the NCI. In this 
study delayed remission induction and duration 
of remission both maintained and unmain- 



NATIONAL CANCER INSTITUTE 



35 



tained have been determined. The study has 
established that a vinca alkaloid is as effective 
as an alkylating agent in the treatment of pa- 
tients with lymphosarcoma and Hodgkin's Dis- 
ease. 

Combination Therapy in Solid Tumors 

In patients with solid tumors, a study has 
been completed in which a combination of 
drugs was used to determine the relative rate 
of remission as well as toxicity. Cyclophospha- 
mide, Oncovin, methotrexate, and 5-fluorouracil 
were given at almost full dosage for 5 day pe- 
riods with a rest period of 14 days between 
courses. This combination was well tolerated 
and toxicity was not excessive. There was a 
significant incidence of tumor responses in pa- 
tients with carcinoma of the breast and with 
testicular tumors. 

This study is being continued in an attempt 
to determine the relative value of the concur- 
rent use of antibiotics on hematologic and gas- 
trointestinal toxicities. One group receives oral 
non-absorbable antibiotics and the second 
group acts as a control. Patients are random- 
ized in the usual fashion. To date; 20 patients 
have been studied in this program with 11 hav- 
ing received antibiotic therapy and 9 in the 
control group. Initial data suggest that the du- 
ration of fever and the incidence of infection in 
the treated gi'oup are somewhat lower but the 
differences are not statistically significant at 
this point. 

Chronic Myelocytic Leukemia 

Intensive studies in patients with chronic 
myelocytic leukemia continue. These encom- 
pass all aspects of the disease. About 85% of 
the patients with this clinical diagnosis will 
have the Philadelphia chromosome. It also ap- 
pears that those who do not have the abnormal 
chromosome have a shorter survival, that is, 18 
months compared to 45 months in the individ- 
uals who are Ph^ positive. 

A major effort is being made to control the 
blastic crisis of patients with chronic myelo- 
cytic leukemia. Combination drug treatment 
with prednisone, methotrexate, and 6-mercap- 
topurine (PAP) has been used. Three of five pa- 
tients who received one course of drug treat- 



ment have gone into remission. However, these 
remissions have been of very short duration 
lasting from 1 week to 3 months. Treatment in 
this stage of chronic myelocytic leukemia is 
made more difficult because it is not possible to 
make a definitive diagnosis early. Hence, the 
possibility remains that intensive therapy is 
being started too late in the course of the com- 
plication and that success would be more likely 
if therapy M^ere begun at an earlier time. Leu- 
kocyte kinetic studies with tritiated thymidine 
may be of value in arriving at an earlier diag- 
nosis. 

Acute Leultemia 

The major advance in chemotherapy in acute 
leukemia during the last year was the develop- 
ment of what appears to be effective chemo- 
therapy for adult leukemia. Remission induction 
was achieved in 19 of 24 patients with acute 
myelocytic leukemia over the age of 20. This 
was accomplished with the POMP combination 
chemotherapy program (prednisone, Oncovin, 
methotrexate, 6-mercaptopurine) . 

In children with acute lymphocytic leukemia 
it has been shown that intensive therapy early 
in remission can significantly prolong the dura- 
tion of unmaintained remission. In addition, 
better techniques for maintaining remjssion 
once achieved have been evolved using inter- 
mittent methotrexate therapy. A study of the 
importance of the duration of treatment of pa- 
tients in remission should permit the design of 
remission maintenance therapy which will very 
greatly prolong the duration of the initial re- 
mission. 

Multiple Myeloma 

A study has been completed using Cyclophos- 
phamide in multiple myeloma. It appears to be 
as effective as phenylalanine mustard. Metho- 
trexate and 6-thioguanine have also been tried 
but were found to be ineffective in this disease. 

A cooperative effort with the Metabolism 
Branch and the NIAID and NIDR has been 
completed in which fluoride was utilized in the 
treatment of bone disease in patients with mul- 
tiple myeloma. It appears that fluoride will cor- 
rect the negative calcium balance in these pa- 
tients with incorporation of the halogen into 



36 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



bone as determined by chemical analysis of 
biopsy specimens. Studies of the crystalline 
structure of the bone as well as its histology 
are now underway. 

New Agents 

During the year several new agents have un- 
dergone initial evaluation. The most promising 
of these has been methyl isopropyl carbamyl 
benzyl hydrazine (M.I.H.) . This drug shows no 
cross resistance to any known chemotherapeu- 
tic agent and has induced a 50-75% remission 
rate in Hodgkin's Disease. 

The effect of cytosine arabinoside in patients 
with multiple myeloma is being evaluated. This 
drug has also been tested in a relatively small 
number of patients with chronic myelocytic 
leukemia but does not appear to be particularly 
promising at this time. Early studies in acute 
leukemia suggest that it may have some value 
both in remission induction and maintenance. 

Leukocyte Dynamics in Leukemia 

Studies of the leukocyte kinetics in patients 
with leukemia, primarily chronic myelocytic 
leukemia continue. Although the analysis is not 
yet complete it appears that in these patients, 
in contrast to normal individuals, the cells are 
synthesizing DNA in the bone marrow as v/ell 
as in the peripheral blood and that the cells are 
released from the bone marrow and /or spleen 
into the peripheral blood. These cells then re- 
enter the marrow and spleen. Cell division in 
the bone marrow appears to progress only to a 
certain point and does not appear to continue 
in the peripheral blood. The myelocyte is the 
predominant cell involved. If any significant 
number of this large group of cells in this dis- 
ease were to divide, many labelled mature 
granulocytes would appear in the peripheral 
blood at some time during the period of obser- 
vation. However, this does not seem to be the 
case as determined from the analysis of the ra- 
dioautographs. The studies have also suggested 
that cells in chronic myelocytic leukemia sur- 
vive or recirculate in the peripheral blood with 
a half-time between 60 to 90 hours. Similar 
studies in patients with acute myelocytic leuke- 
mia have suggested that the intravascular sur- 
vival (half-time) is approximately 12 hours. Of 



particular interest is the observation that pa- 
tients in the blastic crisis of chronic myelocytic 
leukemia have curves which appear to be iden- 
tical with those of patients with acute leukemia 
and in marked contrast to the curves seen in 
patients with chronic myelocytic leukemia. 
This observation may be important in the diag- 
nosis of this particular phase of the disease. 

Leukocyte and Platelet Transfusion 

The white blood cell and platelet transfusion 
program continues to constitute a major effort 
in the Medicine Branch. The availability of a 
plasmapheresis unit in the acute leukemia area 
has become a very valuable resource for the 
collection of leukocytes, the improvement of 
platelet concentrates, and for the collection of 
plasma from patients. 

A more effective technique has been devised 
for the preparation of platelet concentrates in 
acidified plasma which has virtually overcome 
the problem of preparation of good platelet 
concentrates. This new technique results in a 
preparation of platelet concentrates which are 
equal in efficacy to the platelet-rich plasma 
preparations. 

Granulocyte transfusions as a supportive 
measure are being used with increasing fre- 
quency. Previous studies with transfusions of 
granulocytes obtained from patients with 
chronic myelocytic leukemia have suggested 
that this type of replacement therapy may be 
valuable as a supportive measure during the 
marked leukopenic stage resulting from inten- 
sive chemotherapy. However, during the past 
year in addition to these studies, high dose nor- 
mal white cell transfusions have been initiated 
using Dextran separated granulocytes from 
freshly drawn units of normal blood. Prelimi- 
nary results suggest that the percent recovery 
normal granulocytes is similar to that obtained 
with the leukemic granulocytes. Effectiveness 
of homologous normal granulocytes in control- 
ling infection in leukopenic individuals is diffi- 
cult to evaluate primarily because of the prob- 
lems in obtaining sufficient numbers of normal 
cells. As soon as these become available in 
quantity, it will be important to perform a 
comparative study with leukemic granulocytes 
to establish relative effectiveness. 



NATIONAL CANCER INSTITUTE 



37 



It had been hoped that with the development 
of a continuous white blood cell separator that 
there would be less reliance on the cells from 
patients with chronic myelocytic leukemia for 
transfusion purposes. However, although the 
separation of leukocytes from bank blood has 
been quite satisfactory, the yield on an in vivo 
basis has been poor. Intensive study of the rea- 
sons for this variance between fresh blood and 
bank blood has revealed that the difference is 
probably due to a greater tendency toward rou- 
leaux formation in stored blood. Additional 
studies indicate that the densities of leukocytes 
and erythrocytes overlap. Accordingly, it has 
become quite obvious that the separation of 
these two types of cells will be feasible only by 
the use of a medically acceptable additive. This 
study is now proceeding and the early results 
appear to be promising. 

In the course of the development of the leu- 
kocyte separator, other uses for the machine 
have suggested themselves. With relatively 
minor modification of the present design it is 
now possible to procure large numbers of plate- 
lets and lymphocytes. Separation of the plasma 
and the cellular elements of blood in vivo on a 
continuous basis as well as in vitro is quite 
practical. Finally, a modified version of the 
present machine can be used for the deglycerol- 
ization of frozen blood in less time than by any 
other known method. 

"Life Island" 

A development of potential major importance 
during the last year appears to be the intro- 
duction of the "Life Island" reverse isolation 
technique. Four patients have been entered but 
only three patients can be evaluated including 
one individual with acute lymphocytic leuke- 
mia, one with acute myelocytic leukemia, and 
one with a testicular carcinoma and widespread 
metastases. All received amounts of chemo- 
therapy which under ordinary circumstances 
might have resulted in life threatening toxicity. 
However, in the "Life Island" toxicity appeared 
to be relatively moderate. Leukopenia was 
severe in all three individuals but there were 
no infections, buccal ulcerations, or other 
toxic manifestations. Response to therapy was 
quite satisfactory in the first two patients. The 



third patient, who is still in the unit at this 
time, has shown complete tumor regression. 
These preliminury studies suggest that it may 
now be possible to administer chemotherapy at 
dosages far in excess of what is ordinarily given 
at a risk of comparatively moderate toxicity. 

Biochemistry of Leukemic Leukocytes and Ma- 
lignant Cells 

The biochemistry of leukemic leukocytes and 
malignant cells in general continues to be an 
area of increasing emphasis within the Medi- 
cine Branch. 

It has been suggested by a number of individ- 
uals that thymidine may exert a controlling or 
regulatory influence on DNA synthesis in 
leukemic cells. Accordingly, the effect of thymi- 
dine on nucleic acid and protein synthesis in 
the leukocytes from patients with chronic and 
acute leukemia has been studied intensively 
during the past year. It appears that the inhi- 
bition of DNA synthesis is dependent on the 
constant presence of thymidine in the medium 
but is reversed by its removal or by the addi- 
tion of deoxycytidine. The importance of pre- 
formed or exogenous thymidine in DNA syn- 
thesis in leukemic cells has not yet been clearly 
established. However these studies have indi- 
cated that low concentrations of thymidine in- 
hibit DNA synthesis. It is intriguing to speculate 
on this since thymidine in significant quan- 
tities is readily available from dietary sources 
as well as from endogenous pools by catabolism 
of DNA released from disintegrating cells. 

Further studies with other inhibitory sub- 
stances which have been isolated from leuke- 
mic white cells continue. Although the evidence 
is not yet definitive it may be that thymidine 
phosphorj^lase, described last year, may be the 
inhibitory substance responsible for the indi- 
rect effects of splenic irradiation, remissions 
obtained in leukemia by hypertransfusion, etc. 

In addition to studies of DNA synthesis in 
human leukemic cells, RNA synthesis in lym- 
phocytes of patients with chronic lymphocytic 
leukemia has been investigated. These cells are 
capable of synthesizing large amounts of rela- 
tively stable RNA. Using the technique of 
DNA-RNA hybridization one can demonstrate 



38 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



that one-third of the RNA is rapidly synthe- 
sized and that it can hybridize with homolo- 
gous DNA. In addition, a large portion of this 
RNA resembles ribosomal RNA. These com- 
ponents are present even after 24 hours of in- 
cubation, a situation quite different from other 
mammalian cell lines. 

Virus Studies 

In close collaboration with the Laboratory of 
Viral Oncology the isolation and identification 
of the virus-like particles found in patients 
with leukemia is being pursued. In addition, 
the Medicine Branch is continuing to assist the 
Laboratory of Viral Oncology in the immunolog- 
ical study of the relationship between these 
virus-like particles and the presence of leuke- 
mia. 

A finding of potentially great importance but 
is still not definitive is the observation that 
about 1/3 of the patients with leukemia had par- 
ticles in their urine on electron microscopy 
which resembled those seen in plasma of the 
patients with leukemia by Dalton and his colla- 
borators. 

Conclusion 

It is apparent from the foregoing review of 
the activities of the Medicine Branch that sen- 
ior members of the branch have become heavily 
involved in research activities which are vital 
to a comprehensive study and understanding 
of the etiology, mechanisms, and treatment 
of leukemia and solid tumors in man. These 
include: 1) studies of immune competence in ma- 
lignant disease and the effect of drugs, 2) cyto- 
genetics, 3) physiology and dynamics in leuke- 
mia, 4) biochemistry of leukemic leukocytes 
and malignant cells, 5) platelet and leukocyte 
antibodies, 6) bone marrow transplantation, 

(a) in support of patients with malignancies 
who have undergone intensive treatment, 

(b) in leukemia after "ablation" therapy, 7) in 
collaboration with other groups, studies of the 
incidence of virus-like particles, PPLO, and cyto- 
megalic inclusion virus in leukemia, 8) perfu- 
sion studies for (a) bone protection and (b) 
for the investigation of tumor blood flow for 
the possibility of enhancing drug exposure. 



With all these areas under investigation it 
might be of value from an administrative 
standpoint to consider a creation of additional 
sections to include cytology — cytogenetics, 
biochemistry (leukemia, virus) and perfusion. 
At least 2 additional senior individuals are 
needed — a research cytogeneticist and a 
biochemist with a predominant interest in leu- 
kocyte biochemistry, control mechanisms, and 
viral effects. These would fill voids which now 
exist. 

Having these tremendous resources, the Med- 
icine Branch is in a highly favorable position 
to make major contributions in leukemia and 
certain of the malignancies. It appears that of 
all the malignancies, leukemia should be the 
first disease amenable to a solution and cure. 
This disease (s) is being approached from all 
angles — etiologic, physiologic, biochemical and 
therapeutic. In the solid tumors, it has become 
obvious that greater emphasis should now be 
placed on "disease orientation" rather than on 
drug orientation. The clinical drug evaluation 
program has provided a number of drugs 
which singly or in combination result in signifi- 
cant tumor regression and remission rates in 
three diseases: 1) the lymphomas, particularly 
Hodgkin's Disease, 2) testicular tumors, 3) 
carcinoma of the breast. It is these, in my opin- 
ion, to which our attention should now be di- 
rected. With the very early promising results 
in the "Life Island" and with the support of 
blood products, "maximum" antitumor therapy 
may now be possible without prohibitive tox- 
icity. 

LABORATORIES OF VIRAL CARCINOGEN- 
ESIS AND VIRAL ONCOLOGY 

During the past fiscal year activity was in- 
creased in the tumor-virus program of NCI 
and the large Laboratory of Viral Oncology 
was split into two laboratories under the newly 
created ofiice of Associate Scientific Director 
for Viral Oncology. Dr. Bryan was appointed 
Associate Scientific Director but also remained 
as Chief of the Laboratory of Viral Oncology. 
Dr. Dalton was appointed Chief of the second 
laboratory, designated as the Laboratory of Vi- 
ral Carcinogenesis. Scientific members of both 
laboratories continue to work together as a 



NATIONAL CANCER INSTITUTE 



39 



single functional group with respect to partici- 
pation in the collaborative human cancer-virus 
program of the Institute, and, during the last 
half of the year, several of them accepted re- 
sponsibility for the administration of segments 
of the newly created Special Virus-Cancer- 
Leukemia Program of the National Cancer In- 
stitute. Doctors Dalton, Fink, Manaker and 
Moloney are serving as Program Segment 
Chairmen or co-Chairmen, and Dr. Rauscher is 
the scientist member of the Management Team 
of the Special Program. Doctors P. Mora, T. 
O'Connor and S. Stewart are members of Seg- 
ment Working Groups. 

Within the two laboratories the spectrum of 
scientific disciplines and special skills ranges 
all the way from the molecular level to the level 
of the intact animal host, including man. Al- 
though emphasis on research is directed pri- 
marily toward viruses in relation to cancer, the 
research effort also includes studies on basic 
problems underlying solutions of tumor virus 
problems. These include: the interactions of 
macromolecules in isolated and interpretable in 
vitro systems; the ultrastructure of cells, in- 
cluding normal and non-viral cancer cells as 
well as cells of virus induced cancers ; immuno- 
logical phenomena in relation to cancer; basic 
virology, including viral ultrastructure and 
virus-host interactions in vitro and in vivo ; and 
the development and refinement of technology 
within these areas. 

In the Laboratory of Viral Carcinogenesis 
Dr. Sarah Stewart has succeeded in establish- 
ing a continuous line of malignant cells from a 
biopsy of Burkitt lymphoma in a 7-year-old Ni- 
gerian male who was admitted to the Clinical 
Center. In collaboration with Dr. Stewart the 
cells in tissue culture were shown by Dr, Dalton 
to carry a virus which in electron-microscopic 
morphology resembles the herpes viruses and 
the Lucke frog kidney carcinoma virus. The' 
virus is morphologically the same as that 
found in 3 different established cell lines de- 
rived by Epstein (London) from as many differ- 
ent cases of Burkitt lymphoma. This brings to 
4 the number of successfully established cell 
lines of this tumor, and each of the 4 lines car- 
ries the same type of viral agent. Studies by 
Epstein on his cell lines have shown the virus 
to differ from herpes virus in both immunologi- 



cal and biological properties. The virus is now 
considered to be a serious candidate for an 
etiological agent of Burkitt lymphoma. Addi- 
tional studies by Dr. Stewart succeeded in es- 
tablishing conditions under which a viral agent 
from the Burkitt cell cultures, presumably the 
agent identified with the electron microscope, 
can be passaged serially in hamsters. This 
work is still in an early phase but if repeatable 
with the other 3 candidate virus isolates, this 
could provide the basis for rapid animal bioas- 
says and thereby revolutionize the problem of 
determining etiological significance of the Bur- 
kitt-associated-virus. Other studies of Dr. Stew- 
art aimed at finding susceptible hosts for pos- 
sible agents recoverable from human leukemia 
patients led to the discovery of a new canine 
virus. Although "herpes-like" in electron mi- 
croscopic structure, it differs from herpes as 
well as from previously known canine viruses. 
The virus is transmissible in suckling pups in 
which it produces lymphadenopathy and hem- 
orrhagic lesions. There is evidence also that 
this agent is responsible for some cases of ca- 
nine abortion. The electron microscopic studies 
were carried out by Dr. David-Ferreira, in col- 
laboration with Dr. Stewart. Furher investiga- 
tions of the "factor" previously reported by 
Dr. Stewart which enabled standard strains of 
Rous sarcoma virus (a chicken virus) to cross 
species into mammals (hamsters) have indicat- 
ed that it probably is a non-specific enhancing 
factor, and not a unique substance which en- 
dows a new qualitative, species-crossing prop- 
erty. This is further indicated by the finding 
of Dr. Rabotti that very high doses of all 
strains of sarcoma virus thus far tested are ca- 
pable of inducing tumors in hamsters without 
any added enhancing factor. It should be point- 
ed out that an intensive research effort (under 
contract) directed toward the isolation and 
identification of this "factor" led to the discov- 
ery that a known chemical substance, dimethyl- 
sulfoxide (introduced as one of the "controls"), 
also enhanced the biological activity of Rous 
sarcoma virus. The inclusion of dimethylsul- 
foxide in the suspending fluid was a crucial 
part of the procedure developed by Dr. Stewart 
for successful passage of the Burkitt-associat- 
ed-virus in hamsters. 



40 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Dr. Manaker has succeeded in developing a 
continuous line of malignant cells from an 
adult male who died of acute lymphotic leuke- 
mia about one month after the expiration of 
his wife with the same disease. A report just 
received from the contract supported electron 
microscopy collaborative group at the Pfizer 
Company (May wood, N.J.) indicates that this 
tissue culture cell line carries a "herpes-like" 
viral particle similar to that carried by the 
Burkitt lymphoma cells as well as by two cell 
lines derived from human myelocytic leukemia 
(Grace, Roswell Park) . In studies on an avian 
tumor virus isolated from a methylcholanth- 
rene-induced tumor of a Japanese quail {Co- 
turnix sarcoma virus (CSV), by Reyniers, 
Tampa) , Dr. Manaker has succeeded in propa- 
gating the agent in quail tissue cultures and 
has determined some of its properties. The 
virus is highly stable and appears not to be of 
the defective type as is the Rous sarcoma virus 
of chickens. The tissue culture-grown quail 
virus produces tumors in chickens which retro- 
gress after about three weeks in this host. 
Further studies on biological and immunologi- 
cal properties are in progress. Studies on sev- 
eral established cell lines of mouse myeloepith- 
elioma by Dr. Manaker in collaboration with 
Doctors Rabotti and David-Ferreira have 
shown that this mouse tumor carries immature 
"type C" virus particles, but biological tests 
have failed to show transmissibility of this 
neoplasm by filtrates. A small but significant 
portion of the inoculated mice develop leuke- 
mia, indicating that the tumor cells may be 
carriers of a mouse leukemia virus. Dr. Man- 
aker is leading an intensive collaborative effort 
involving both intramural and extramural 
(contract supported) scientists of various dis- 
ciplines in attempts to increase the production 
of Burkitt-associated-virus and isolate and pu- 
rify it from lymphoma cells in tissue culture. 

Dr. Rabotti has carried out quantitative stud- 
ies on the induction of brain tumors in ham- 
sters by Rous sarcoma virus (a chicken virus), 
and found that standard strains of this virus, 
previously thought incapable of crossing broad 
species barriers, can induce brain tumors 
in hamsters if the inoculated dose is high 
enough. The oncogenic dose of standard Rous 
sarcoma virus was thus found to be 10^ PFU, 



whereas other strains such as the Schmidt- 
Ruppin strain previously shown to cross spe- 
cies readily requires only about 10^ PFU. Ex- 
ploring this quantitative finding further and 
using very large doses of Rous sarcoma virus, 
Dr. Rabotti has succeeded in inducing brain tu- 
mors in guinea pigs, rabbits and dogs. The tu- 
mors in guinea pigs were leptomeningeal sar- 
comas, only, whereas in rabbits and dogs both 
gliomas and leptomeningeal sarcomas were in- 
duced. Both Schmidt-Ruppin and standard 
strains of the virus were effective provided 
"oncogenic doses" were used. This technique 
for rapidly inducing tumors in dogs (within a 
few weeks) is of considerable importance to 
cancer chemotherapy and related pharmacolo- 
gical investigations because, up to now, there 
has been no available experimental tumor of 
large animals for use in pharmacological stud- 
ies, prior to trial of candidate therapeutic 
agents in man. The brain studies in dogs were 
in collaboration with Dr. A. Grove (NINDB). 
Dr. Rabotti is also collaborating with Dr. D. 
Rail, LCP, in developing the use of dog men- 
ingeal tumors as a test system for trials of chem- 
otherapeutic agents selected for potential 
effectiveness in the treatment of meningeal 
leukemia. 

Dr. Dalton and other electronmicroscopists 
of his group continue to provide the "eyes" in 
the search for viral agents associated with hu- 
man neoplasia, particularly leukemia, as well 
as participating in mutidisciplinary investiga- 
tions on both human candidate tumor viruses 
and viruses of known animal viral neoplasias. 
As already mentioned. Dr. Dalton has studied 
cells of the Burkitt lymphoma line (SLI) estab- 
lished by Dr. Stewart and has observed 
"herpes-like" viral particles similar to those 
reported by Epstein in his cell lines (EBl, EB2 
and EB3). Dr. Manaker is studying EBl and 
EB2 cells obtained from Dr. Epstein. Dr. Dal- 
ton in collaboration with Dr. Manaker has ob- 
served "herpes-like" particles in both cell lines, 
thus confirming the work of Epstein. Also, he 
has confirmed the presence of similar particles 
in one of the human myeloid leukemia cell lines 
established by Grace. In collaboration with 
Doctors Sanford and Evans, LB, Dr. Dalton 
has found both A and C type viral particles in 
established mouse cell lines in tissue culture. 



NATIONAL CANCER INSTITUTE 



41 



The C type particles are present when the cells 
are grown in media containing horse serum, 
but not in parallel lines grown in chemically 
defined media containing no horse serum. 
These observations held for Earle "L" cells and 
Dr. Sanford's "High" line, both of which were 
established initially by explanting normal 
mouse fibroblasts, but eventually underwent 
"spontaneous" transformation to malignancy 
in vitro. In a survey of mammary tumors sup- 
plied by Doctors Andervont, Heston and 
Deringer, LB, mature type B particles, as well 
as A type particles, were found in all but those 
tumors arising in EIII and Deringer C3He 
mice. In these two instances A type particles, 
only, were found. Dr. Dalton has also identified 
as type C the virus associated with the mouse 
sarcoma recently discovered by Dr. Moloney. 
The particles were indistinguishable morphol- 
ogically from those of the Moloney and other 
mouse leukemia viruses. 

Dr. David-Ferreira completed and published 
his thorough electronmicroscopic study of the 
mouse hepatitis virus (A-59) discovered and 
previously reported by Dr. Manaker. Dr. Man- 
aker collaborated with Dr. David Ferreira in 
this investigation. As already mentioned, Dr. 
David-Ferreira collaborated with Dr. Stewart 
in determining the morphology of the new ca- 
nine virus which she discovered. 

Dr. Bucciai'elli, a guest v/orker from Peru- 
gia, Italj^, is collaborating with Dr. Rabotti in 
the study of brain tumors in dogs induced with 
the Rous sarcoma virus. Dr. Dietert, who re- 
cently joined Dr. Dalton's staflf, has begun a ra- 
dio-autographic study aimed at following the 
movement of Moloney lymphocytic leukemia 
virus RNA from its entrance into the cell to its 
replication as newly formed virus particles. 

Dr. Douglas Anderson, in collaboration with 
Doctors Manaker and Barile, DBS, has com- 
pleted an extensive electron microscopic study 
of two strains of mycoplasma. His results have 
greatly increased our capability of distinguish- 
ing organisms of mycoplasma from virus par- 
ticles in thin section preparations. 

Mrs. E. Mitchell continues her studies of ul- 
tracentrifuge pellet material derived from 
plasma obtained by plasmaphoresis from leuke- 
mia patients entering the Clinical Center. Of 
96 specimens examined with thin-section elec- 



tron microscopy 19 were positive for virus-like 
particles and 6 were questionable. The larger 
percent (about 20) of definitely positive speci- 
mens as compared with the study reported last 
year (about 12% positive) is associated with 
the larger samples obtained by plasmaphoresis 
and the increased amount of plasma contribut- 
ing to the pellet specimen in each case. 

In the Laboratory of Viral Oncology Dr. Mo- 
loney has discovered a new mouse tumor virus 
which produces solid tumors (rabdomyosar- 
coma) at the site of inoculation within two 
weeks after virus administration. The agent 
came to light in a routine biological test of a 
highly concentrated preparation of Moloney 
mouse leukemia virus. Certain of the recipient 
mice inoculated Avhen newborn developed tu- 
mors at the site of inoculation. The solid tu- 
mors can be reproduced in serial passages of 
the virus derived either from extracts of tumor 
tissue or from the plasma of tumor-bearing 
mice. As already mentioned, Dr. Dalton has 
found this agent to be indistinguishable from 
mouse leukemia virus morphologically. The 
agent withstands repeated freezing and thaw- 
ing and has been found fully active after sto- 
rage for at least 4 months at dry ice tempera- 
ture (— 76°C) . Young mice up to 14 days of age 
(the oldest yet tested) are susceptible to tumor 
induction by this virus and tumors have been 
produced in 100 percent of the mice inoculated 
among 6 different strains and 2 hybrid crosses. 
The average latent period varied from 9 to 16 
days among the different strains and the time 
to death- with-tumor from 21 to 42 days. A 
brief note was recently published by Harvey 
(Nature, No. 4963, 1964) of similar results ob- 
tained in studies carried out in England. Solid 
tumors arising at the site of inoculation of 
strong doses of Moloney mouse leukemia virus 
were said to be transmissible with cell-free 
fractions, but further details were not given. 
Whether the virus that causes solid tumors is a 
varient of the Moloney leukemia virus or a dif- 
ferent agent that has been picked up and now 
"contaminates" Moloney virus preparations re- 
mains to be determined. The new agent is not 
neutralized by concentrations of specific anti- 
serum which completely inactivate the Moloney 
mouse leukemia virus, but a delay in the latent 
period of tumors arising after incubation of 



42 



ANNUAL REVIEW OF INTKAMURAL RESEARCH 



mixtures of the virus and anti-Moloney mouse 
leukemia virus antiserum suggests that the two 
viruses have some antigenic similarity. In fur- 
ther studies of the mouse leukemia virus which 
he isolated from sarcoma 37, Dr. Moloney has 
increased the biological activity by selected 
serial passage and has demonstrated that, as 
with the Rous sarcoma virus, the amount of 
virus recoverable from diseased animals is 
quantitatively related to the infecting dose of 
virus. Dr. Moloney has also developed a more 
rapid bioassay for his mouse leukemia virus, 
using the weights of spleens 28 days following 
virus inoculation. 

Dr. Gordon Theilen (on sabbatical leave 
from the University of California) has been 
studying the avian virus of Twiehaus which 
was originally isolated from a spontaneously 
diseased turkey, but which also causes a malig- 
nant endotheliosis in chickens. He has found 
that similar disease is induced in Japanese 
quail, and quantitative dose-response relation- 
ships of this agent have been studied in chicks. 
Dr. Thielen has collaborated with Dr. Zeigel in 
electron microscopic studies of the Twiehaus 
virus, and with Dr. Dalton in a search for viral 
particles in specimens from leukemic cows. 
Particles of the C type have been seen in lymph 
nodes from one leukemic cow. Dr. Thielen has 
also participated in a collaborative study of an- 
imals, particularly dogs, associated with cases 
of human leukemia. About 10 plasma speci- 
mens obtained by him are now being studied by 
electron microscopy. 

Dr. Ruth Merwin has obtained additional 
evidence that the diffusion chamber method de- 
veloped by her for the detection of small 
amounts of tumor virus is a valuable new 
procedure for bringing to light smaller 
amounts of such viruses than can be detected 
by conventional extraction and inoculation 
procedures. As previous^ reported, a virus iso- 
lated by this method from sarcoma 37 produced 
both leukemia and "bone lesions" resembling 
osteopetrosis of chickens. In further studies 
Dr. Merwin has shown that the Moloney mouse 
leukemia virus, likewise isolated from sarcoma 
37, also induced "bone lesions." It is probable 
that Dr. Merwin's isolate is the same as Dr. 
Moloney's. Experiments to determine this are 
in progress. The induction of bone lesions by 



the Moloney virus had not previously been 
detected. In continuation of her studies on the 
induction of plasma cell tumors in BALB/c 
mice by intraperitoneal implantation of plex- 
iglas and other membraneous discs. Dr. Mer- 
win has observed hyperplastic plasma cells dis- 
tributed over the peritoneal surfaces as early 
as 3 months after implantation of the discs. 
Grossly diagnosable plasma cell tumors did not 
appear until 6 months after implantation of 
the discs. 

Dr. Rauscher has carried out quantitative 
studies on the growth curve of his mouse leuke- 
mia virus in BALB/c mice. Virus could not be 
recovered through the 3rd to 4th day after 
virus inoculation, but beginning with the 5th 
day virus was present in both plasma and 
spleen tissue in relatively large amounts. It in- 
creased with time at a regular rate until a peak 
was reached which was dependent on the dose 
of virus inoculated. The time to peak was also a 
function of initiating dose, being 21 days with 
the 10"'' dilution of virus (about 5 ED50). At 
this lower initiating dose level virus could not 
be demonstrated in plasma or spleen in about 
14 of the animals sacrificed 48 days after inoc- 
ulation. The results are comparable to those 
that have been obtained with Rous sarcoma 
virus and indicate that, as in Rous sarcoma, 
the neoplasia is viral dependent. Similar stud- 
ies in rats and in C57BL mice showed the 
growth curve of virus to be comparable, but 
the curve was more extended in time in rats. In 
this host the eclipse phase in Avhich no virus 
could be detected was 18 to 22 days. The re- 
sponse of C57BL mice was comparable to that 
of BALB/c during the initial stages, but in 
C57BL hosts the virus declined progressively 
after reaching its peak, in contrast to its con- 
tinued high level through life in the BALB/c 
strain. A characteristic of the Rauscher virus 
which distinguishes it from other mouse leuke- 
mia viruses is its ability to stimulate erythro- 
poiesis and induce marked splenomegaly within 
10 to 14 days following virus inoculation. Lym- 
phocytic leukemia appears later, after 2 or 3 
months. The initial manifestations of the bi- 
phasic disease do not appear in rats. After pas- 
sage in rats the recovered virus also fails to 
induce erythropoiesis and splenomegaly when 



NATIONAL CANCER INSTITUTE 



43 



inoculated back into mice, although leukemo- 
genic activity is retained. These results suggest 
the possibility that the original virus material 
isolated by Dr. Rauscher may have consisted of 
2 different agents. This is supported by addi- 
tional studies of Dr. Rauscher in which the 
virus passaged in tissue culture lost its ability 
to induce erythropoiesis and splenomegaly 
when inoculated back into BALB/c mice. How- 
ever, all attempts to isolate and propagate 
the erythropoietic factor separate from the 
leukemogenic agent have failed thus far. 
Furthermore, virus recovered from leukemic 
rats of the 10th serial passage in rats unexpec- 
tedly induced marked erythropoiesis in addi- 
tion to leukemia when inoculated back into 
BALB/c mice. The question of 2 agents, or mod- 
ulations involving a single agent, cannot be 
resolved at this time. Dr. Rauscher has contin- 
ued his collaboration with scientists of other 
disciplines in extensive studies on the chemical, 
immunological, biophysical and electron mi- 
immunological, biophysical and electron mi- 
croscopic characteristics of his virus isolate. 
He has provided the essential, parallel, quanti- 
tative biological and virological components of 
the joint investigations. 

Dr. Zeigel in collaboration with Doctors 
Rauscher, Fink and Tyndall (Oak Ridge Na- 
tional Laboratory) has carried out further stu- 
dies on the Rauscher virus. A significant 
amount of virus is produced by budding from 
cell membranes and evidence has been found 
that there is a concurrent phagocytosis of viral 
particles by the same cells. This suggests a 
"dynamic equilibrium" with regard to free 
virus in culture fluid (i.e., virus titer) . A syste- 
matic electron microscopic study is being made 
by Dr. Zeigel of various tumors of BALB/c 
mice. "A" type particles which bud from mo- 
dified endoplasmic reticulum have been found 
in all tumors examined thus far. These include 
an ascites form of Rauscher leukemia cells, a 
spontaneous solid tumor which arose in mice 
inoculated with Rauscher virus, two epithe- 
liomas, both primary and transplanted plasma 
cell tumors, a spontaneous mammary tumor, 
two myoepitheliomas, a methylcholanthrene in- 
duced epithelioma and a spontaneous lympho- 
cytic tumor. In addition to the Rauscher virus 
lesions, several of the other tumors also con- 



tained "C" type particles indistinguishable 
from those associated with mouse leukemia. 
Studies on the CELO virus (a chicken virus) 
have confirmed its structure as an adeno-like 
virus possessing 252 capsomeres and Dr. Zeigel 
now regards it as probably identical with the 
Gal virus (Gallus adeno-like virus) . These stud- 
ies were carried out with the collaboration of 
Dr. Sarma (formerly of NIAID). In collabora- 
tion with Dr. Theilen, Dr. Zeigel has studied 
the Twiehaus virus and found that the par- 
ticles are not identical with other known 
strains of avian lymphomatosis, myeloblas- 
tosis, and erythroblastosis viruses in electron 
microscopic morphology. He is continuing his 
basic studies on normal development of thymic, 
liver, and pancreatic cells and is collaborating 
in the Institute program on human leukemia 
through periodic examinations of plasma speci- 
mens of monkeys inoculated, when newborn, 
with candidate agents derived from human 
leukemia. 

The development of fluorescent antibody 
techniques applicable to mouse leukemia vi- 
ruses and to candidate viruses from human 
leukemia and lymphoma were reported last 
year by Doctors Fink and Malmgren (LP). Dr. 
Fink has carried out additional studies on hu- 
man leukemia using fluorescent antibody 
prepared in both rabbits and monkeys against 
the pellet fraction derived by differential cen- 
trifugation from plasma of leukemic patients 
which had been found by Dr. Dalton to contain 
particles having the ultrastructural character- 
istics of viruses. As controls, comparable ser- 
um reagents were prepared against fractions 
derived from plasma of normal humans and 
from plasma of leukemics containing myco- 
plasma (PPLO) but no virus-like particles. In 
addition, fluorescent antibody was prepared 
against several known strains of PPLO, includ- 
ing strains isolated from leukemic patients. 
Such organisms are frequently present in blood 
and bone marrow specimens from human 
leukemia patients. None of the fluorescent anti- 
body reagents prepared against normal plasma 
fractions or against isolated strains of PPLO 
have been found to react with leukemic cells 
from the buffy coat or bone marrow. On the 
other hand. Dr. Fink continues to obtain posi- 
tive reactions in a significant number of cases 



44 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



when buffy coat and bone marrow cells of hu- 
man leukemic patients are tested with the fluo- 
rescent antibody reagent prepared against se- 
lected, particle-positive, leukemic plasma frac- 
tions. In her studies on a case of Burkitt lym- 
phoma admitted to the Clinical Center, cells 
from the original tumor biopsy were negative 
but later became positive for the specific anti- 
gen after propagation in tissue culture (by Dr. 
Stewart) . As reported previously, two cell lines 
of Burkitt lymphoma established by Epstein 
were found by Dr. Fink to react positively 
with the human leukemic plasma pellet rea- 
gent. Several impression smears of primary 
biopsy material from Burkitt lymphomas were 
sent to Dr. Fink by Dr. Epstein. These were 
also negative. It should be noted also that Dr. 
Dalton has failed to find virus particles on elec- 
tron microscopic examination of primary 
biopsy specimens of Burkitt lymphoma, where- 
as "herpes-like" virus particles have been ob- 
served after the cells are propagated in tissue 
culture. Three additional cases of erythroleuke- 
mia have been studied by Dr. Fink. Bone mar- 
row specimens of two of them were obtained 
from Dr. Holland of the Roswell Park Memo- 
rial Institute and one from Dr. Lee of Maimo- 
mides Hospital, Brooklyn. The cells of all three 
specimens were positive when tested with fluo- 
rescent antibody against the Rauscher mouse 
leukemia virus (which also induces erythro- 
poiesis) . This brings to 4 the number of human 
erythroleukemia cases that have been tested to 
date and all four have been positive with 
Rauscher virus reagent. The specimens were 
also positive with the anti-human-leukemia 
reagent, but less strongly so. No positive re- 
sults were obtained with any of the control 
fluorescent antibody reagents. In collaboration 
with Dr. Karon (MB) a group of 14 selected 
Clinical Center patients have had repeated 
bone marrow examinations. From this longitu- 
dinal study it appears that the reaction with 
the anti-human-leukemia antibody is present 
when the disease is active and is not present 
when the patient is in remission. The immuno- 
fluoi'escent technique as applied to cells of the 
peripheral blood and bone marrow of leukemic 
humans is believed by Dr. Fink to be detecting 
an antigen associated with leukemia. The cor- 
relation between the presence of "virus-like" 



particles in the plasma, and immunofluores- 
cence of leukemic cells from the same patient, 
supports the hypothesis that the "virus-like" 
particles revealed by thin-section electron mi- 
croscopy are associated with the disease. Con- 
siderable evidence has been accumulated which 
tends to rule out the participation of PPLO an- 
tigen in the positive fluorescent antibody reac- 
tions observed in leukemic cells. 

This immunofluorescent technique may 
prove useful as a screening procedure in addi- 
tion to electron microscopy for detecting candi- 
date materials for biological tests in monkeys, 
attempts at tissue culture propagation, etc.; 
and may provide a clinical diagnostic aid to 
predict relapse of patients before relapse ac- 
tually occurs. The emergence of the ability to 
react by immunofluorescence in lymphoma cells 
in tissue culture, concurrently with the visuali- 
zation of virus by electron microscopy, sug- 
gests that in in vitro systems, beyond the host's 
defensive environment, virus may be produced 
in detectable quantity. 

The work in the macromolecular Biology 
Section of the Laboratory of Viral Oncology 
can be summarized in three areas : Macromolec- 
ular interactions; studies of nucleic acids, 
their base sequence "homology" and their role 
in protein sjmthesis in normal tissues and in 
tumors ; and, the nucleic acids of animal tumor 
viruses including their chemistry and electron 
microscopy. 

In macromolecular interaction studies, Dr. 
Shifrin has continued his research on charge 
transfer complexes. The interaction of biologi- 
cal molecules by charge transfer complex for- 
mation has often been suggested but a defini- 
tive study has not yet been carried out on mole- 
cules of biological significance. Dr. Shifrin is 
pursuing a systematic study of a carefully se- 
lected model system pertaining to enzyme-coen- 
zyme and enzyme-substrate interaction, specifi- 
cally, the complex formation between nicotina- 
mide adenine dionucleotide or thionicotinamide 
adenine dinucleotide and certain electron donor 
molecules. He has found a linear relationship 
between the ionization potential of the electron 
donor and the energy of the intramolecular 
charge transfer transition. This linear rela- 
tionship is an empirical demonstration of quan- 
tum mechanical calculations, and it can be used 



NATIONAL CANCER INSTITUTE 



45 



to predict the position of any charge transfer 
band, knowing the ionization potential of a 
donor molecule. What is more important biolog- 
ically, one can predict from the position of a 
charge transfer band the ionization potential 
and, therefore, the exact nature of a donor 
molecule. Dr. Shif rin has extended his study of 
charge transfer bands to molecular complexes 
in the absence of light. His current studies, 
which include collaborative research using elec- 
tron spin resonance spectroscopy, now pertain 
more broadly to the interaction between amino 
acid side chains and the nucleotide bases. For 
the objectives of pursuing studies on the effect 
of changing the acceptor molecule from nico- 
tinamide to thionicotinaraide, and on how this 
change affects the charge transfer transition. 
Dr. Shifrin developed new organic chemical 
synthetic methods for the preparation of cer- 
tain substituted thiocarbamoylpyridinium 
chloride derivatives. His studies provide 
further insight into the nature of the enzyme- 
coenzjTne complexes and the enzyme substrate 
complexes. In addition, they give information 
on the direct interaction between the bases in 
polynucleotides and the various amino acids in 
polypeptides. 

Dr. Mora in collaboration with Dr. Philip 
Person (VA Hospital, Brooklyn, New York) 
found that the oxidized form of cytochrome c 
changes into the reduced form upon adding a 
polycation to the pure cytochrome c. If an ex- 
cess of polyanion is then added, the reduction 
is reversed and the cytochrome c can be recov- 
ered again in the oxidized form. These reduc- 
tions and oxidations can be repeated in cyclic 
fashion and they occur at a characteristic rate 
depending on the poly ions employed. It appears 
that beside the polycation nature of the sub- 
stances which act as reducing agents, these 
also must have hydrophylic characteristics. 
Some of the substituted cationic polyglucose 
derivatives were found to be most effective re- 
ducing agents, although many other physiologi- 
cally occurring cationic biopolymers (histones, 
etc.), are also quite effective. It should be em- 
phasized that the effects of the polyions are ex- 
actly opposite to that which one would expect 
from direct electron transfer. In studies on the 
mechanism of the reduction Drs. Mora and 
Person found that by heating, or by treatment 



with alcohol, the reduction effect of the polyca- 
tions could be reversed. The oxidized form is 
apparently the more stable and more open ter- 
tiary structure. 

Dr. Mora also discovered a similar reduction 
of cytochrome c by certain sugars, for example 
by glucoseamine, galactoseamine or ribose. It is 
of interest that certain other, more reducing, 
sugars and derivatives such as glucose do not 
have this reducing effect on cytochrome c. Both 
the polycation effects and the carbohydrate 
effects apparently represent a mechanism for 
controlling reducibility or oxidizibility of the 
cytochrome c through changing the tertiary 
structure of cytochrome c, thus operating as an 
"allosteric control." These findings allow a 
deeper understanding of oxidative phosphory- 
lation in the mitochrondria. Since in all types 
of cells, animal and plant, the terminal electron 
transport and oxidative phosphorylation are 
meditated by the cytochrome cycle, and the en- 
ergy of ATP is utilized by this process in the 
mitochrondria, it is of importance to determine 
the conditions which control these processes. 

In research on nucleic acids in protein synthe- 
sis, Dr. Luborsky, in collaboration with Drs. 
Sidney Petska and Marshall Nirenberg (NHI), 
studied the sedimentation properties of ribo- 
somes obtained from a streptomycin sensitive 
strain of E. coli. It had been observed pre- 
viously that addition of streptomycin to the 
Nirenberg cell-free amino acid incorporating 
system induces errors in the amino acid incor- 
poration as directed by a messenger RNA. Drs. 
Luborsky and Pestka obtained results which 
indicate that the "ambiguity" errors probably 
arise from the fact that the template activity 
of the messenger RNA in the protein synthesis 
depends on the meditation of an active 70s ri- 
bosomal species to maintain the activated 
aminoacyl-sRNA in a proper position for the 
formation of the peptide bond. , 

Before a DNA duplication or transcription 
process may begin, at least a partial opening of 
the double stranded DNA structure is obliga- 
tory. The influence of polyanion and various 
conditions of pH and temperature on strand 
separation was studied by Dr. Mora on bacteri- 
ophase Tl DNA. He found that the DNA helix 
±5 coil transition occurs at lower pH, at lower 
temperatures, but polyanions do not affect 



46 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



either the pH transition or the temperature 
transition. By inference, it can be deduced that 
the DNA molecules to not influence each other 
in their transition, even when they occur at a 
very high localized concentration, such as in 
the nuclei of cells. Thus, DNA-DNA electrosta- 
tic interactions do not participate in control 
processes of DNA duplication and Dna trans- 
cription. 

Dr. Carl Smith is engaged in long-range stud- 
ies on mammalian messenger RNA, using nu- 
eic acid homology techniques. These tech- 
niques involve the separation of the two nucleic 
acid strands from the double helix (by the 
same heating technique which Dr. Mora stu- 
died on the bacteriophage Tl DNA) into single 
stranded structures, and hybridizing these 
strands with the complementary mRNA which 
selectively adsorbs onto the DNA. The ad- 
sorbed mRNA can then be fractionated away 
from other RNA species such as the ribosomal 
and sRNA. Demonstration and measurement 
of the base sequence complementarity in var- 
ious mrna molecules is thus possible. DNA- 
DNA hybrids can also be similarly worked 
with. The normal rat liver and the Morris 
hepatoma 5123, which is a minimal deviation 
hepatoma were selected by Dr. Smith for his 
studies. He has developed efficient fractiona- 
tion procedures for both the liver and the hepa- 
toma which permit the separation of nuclei 
(where the DNA controlled mRNA synthesis 
occurs) from the cytoplasmic fraction (where 
most of the protein synthesis occurs). To ob- 
tain DNA, he used the extraction procedure of 
Marmur, which he modified for his purpose. 
With these modifications DNA was obtained in 
the 12-14 million molecular weight range. It 
was possible to trap the DNA in an agar gel 
network used to support the single stranded 
DNA during the homology experiments. To ob- 
tain quantitative data the specific radioactivity 
of the nucleic acids must be very high. In 
studying the conditions of injection of p^^ 
orthophosphate, which is then incorporated 
into both DNA and RNA, Dr. Smith studied 
conditions for obtaining liver cytoplasmic RNA 
and also hepatoma cytoplasmic RNA with high 
radioactivity. The RNA preparations were ob- 
tained by cold phenol extraction, both from the 
nucleic fraction and from the cytoplasmic frac- 



tion. These were then hybridized with the unla- 
belled DNA, in separate experiments from 
liver and from hepatoma. Quantitative conclu- 
sions regarding RNA to DNA homology must 
await further experimental development aimed 
at increasing the radioactivity of the mRNA. 
However, the DNA-DNA homology studies 
showed that the liver and hepatoma cells are 
alike in the nucleic acid sequence in their DNA. 

In the studies on tumor-viruses, Dr. Lu- 
borsky, in collaboration with Dr. Bader (FS) 
has devised methods for the production of high 
titer Rous sarcoma virus in moderately large 
amounts in tissue culture. Dr. Luborsky also 
developed methods for concentrating and pu- 
rifying this virus in a stable, infective, form in 
serum-free medium. This was necessary before 
he could proceed with the investigation of the 
biochemical properties of the virus and its 
mode of action in producing neoplastic trans- 
formation of cells. His success involved the use 
of a "cushion" of potassium tartarate solution 
to collect the virus during sedimentation in an 
ultracentrifuge and further fractionation in a 
potassium tartarate density gradient. With 
these methods 46% of the input infectivity was 
recovered with more than a 100-old increase 
in concentration. The infectivity studies were 
carried out by Dr. Bader. One further purifica- 
tion step, involving the use of sephadex G 200, 
was necessary for separating the virus from 
low molecular weight contaminants. By these 
combined methods, Drs. Luborsky and Bader 
also succeeded in preparing highly purified trit- 
um laibelled Rous, and Rous-associated, vi- 
ruses. They also observed during these studies 
that most of the radioactivity could be recov- 
ered if certain plastics, tubes, rather than glass 
tubes, were used for centrifuging the solutions. 
The virus was found to adsorb readily to glass, 
but not to the plastics. Sedimentation proper- 
ties of the Rous virus yielded estimates of 
about 600s for a sedimentation coefficient in ul- 
tracentrifuge studies. This value is in line with 
a 80 m/i diameter, 1.16 g/ml density, particle 
with a total weight of 1.9 x 10^ determined 
by independent measurements. 

Dr. O'Connor has continued his studies in 
collaboration with other members of the Labo- 
ratory of Viral Oncology (Drs. Rauscher, Mo- 



NATIONAL CANCER INSTITUTE 



47 



loney, Fink, and Manaker). Certain physical- 
chemical, biochemical and immunological prop- 
erties of animal leukemia viruses and their 
subviral fractions were investigated, especially 
from the point of view of developing leads that 
will be useful in the search for possible viruses 
that might be involved in some human leuke- 
mia. He employed density gradient studies ex- 
tensively both to characterize these viruses and 
to separate the subviral components obtained 
after various partial degradative treatments. 
In density gradient studies, the Rauscher 
virus, Moloney virus, and Friend virus appear 
to have the same buyoant density 1.16 g/ml, a 
value also obtained by Dr. Luborsky for the 
Rous sarcoma virus. In addition. Dr. O'Connor 
has studied influenza virus (PR8 strain), the 
Newcastle disease virus, and the mouse mam- 
mary tumor agent by centrifuging mainly in 
potassium citrate gradients. Avian myeloblas- 
tosis virus was found to disintegrate in potas- 
sium citrate, but it was successfully banded in 
a sucrose gradient. The density of this virus 
was determined to be 1.15 g/ml. A large 
amount of Dr. O'Connor's effort has been di- 
rected toward the development of a controlled 
degradation procedure for the animal leukemia 
viruses, especially the Rauscher virus. Deter- 
gents, or ether were used for degrading the 
virus, and the subviral components were sepa- 
rated by density gradient centrifugation. The 
lipid rich components have a low density, while 
the lipid poor components, which contain more 
of the nucleoproteins, are heavier. These vari- 
ous components were examined by Dr. de The 
with the electron microscope and are now 
under study by Dr. Fink using fluorescent anti- 
body methods. Similar studies are being ap- 
plied also to the Moloney virus and to the New- 
castle disease virus. These studies on subviral 
components are directed toward the identifica- 
tion of possible human leukemia viruses, as 
well as toward a determination of the relation- 
ships of the various animal leukemia viruses. 
Close liaison is maintained between this proj- 
ect and the research of clinicians and biolo- 
gists working on animal leukemia viruses. Den- 
sity gradient fractionation techniques already 
developed are now being employed on both 
plasma and cell extracts of selected human 
leukemia cases, with the objective of obtaining 



higher quantities of the "virus-like" particles 
present therein. 

The properties of the nucleic acids in the var- 
ious murine leukemia viruses are of impor- 
tance for studies on their mode of action. For 
example, it is necessary to know whether the 
nucleic acid (RNA) is single stranded or 
double stranded, and whether or not it behaves 
as a messenger RNA. Characterization of the 
Rauscher virus nucleic acid was undertaken by 
Dr. Peter Mora. He found that the Rauscher 
virus contains only RNA in detectable amount, 
and that the RNA consists of only the normal 
nucleotides of U, A, G, and C. It is very sensi- 
tive to RNase digestion which causes it to split 
easily during the isolation process into lower 
molecular weight products. This indicates a 
single stranded RNA rather than a double 
stranded form. Single strandedness was 
further indicated by experiments on helix ±5 
coil transition studies by heating and cooling, 
in techniques similar to that carried out by Dr. 
Mora on the DNA of Tl bacteriophage. Since 
these findings indicate that the Rauscher virus 
RNA is single stranded, there is no need to as- 
sume a double stranded RNA structure as a nec- 
essary property of the RNA of cander viruses, 
as has been recently suggested by others. 

Dr. Guy de The has carried out electron mi- 
croscopic studies on certain of the structural 
properties of viruses and on cell-virus relation- 
ships. He has found that ATPase and inosine 
triphosphatase are present in the virus enve- 
lopes of both the Rauscher and Moloney vi- 
ruses. This is similar to the findings with mye- 
loblastosis virus. However, these activities 
were not detected in the membranes of chan- 
nels within the infected cells. The mammary 
tumor agent was also examined by a similar 
technique. Low enzymatic activities were 
detected in this virus and also at the apposed 
cell membrane of acini, where the virus is 
formed by budding. The enzymes were also 
found associated with the Rous sarcoma virus, 
but only when they were produced in vivo; 
virus produced in tissue culture did not contain 
ATPase. In collaboration with Dr. Notkins 
(NIDR), Dr. de The has studied the structure 
of the lactic dehydrogenase virus and found 
that it has an elongated elliptical shape, 40 by 
75 mfi in diameter. They also found the site of 



48 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



replication of this virus to be in the cytoplas- 
mic vacuoles and at the cellular membranes of 
peritoneal macrophages. In collaboration vi^itli 
Drs. Luborsky and O'Connor, respectively. Dr. 
de The is also studying the morphology of the 
Rous sarcoma and Rauscher leukemia viruses 
and their subviral components. 

CLINICAL INVESTIGATIONS 

The accompanying reports from the Clinical 
Branch Chiefs detail the research accomplish- 
ments of each Branch. In addition, attention 
should be called to the following specific items : 
(1) reverse isolation, (2) intensive care, (3) 
recruitment of patients for radiation therapy, 
(4) the creation of the Immunology Branch, 
and (5) the development of an experimental 
surgery for the Surgery Branch. 

While only four patients have been studied 
by reverse isolation in an apparatus known as 
the "Life Island", a term which has been ap- 
plied to this study, the clinical results have 
been striking. It has been possible to maintain 
adults in the chamber for periods of weeks. In 
fact, a patient has been maintained in the "Life 
Island" for eight weeks. It has been possible to 
sterilize effectively the skin and gastrointesti- 
nal tract of these subjects. More important, the 
physicians caring for these patients have been 
able to use doses of chemotherapeutic agents 
considerably in excess of what they would con- 
sider reasonable in other circumstances. This is 
because of the astonishing decrease in toxicity 
to the gastrointestinal tract or at least toxicity 
as manifest by ulcerations in the entire gas- 
trointestinal tract. The prevention of infection 
by fungi and bacteria has also permitted the 
maintenance of patients in the reverse isolation 
apparatus without the attendant danger of 
lethal infection. It is much too early to predict 
the outcome of these studies. However, it is not 
too early to state that the changes in toxicity 
observed are remarkable. It is hoped that this 
procedure will lead to better understanding of 
the role of infection in toxicity and to ways of 
giving patients additional quantities of drugs. 
It should be mentioned that these studies are 
very costly in terms of our personnel resources, 
particularly nursing. 



This past year has also seen the completion 
of an intensive care unit for patients in the 
Surgery Branch. This will now permit all post- 
operative patients to be cared for in a special 
facility designed for this purpose. It will also 
permit the surgeons to care for other patients 
in this facility when the need is indicated. This 
facility has its own nursing staff. 

The past year has witnessed the ability of 
the Radiation Branch to gain referrals of pa- 
tients suitable for radiation therapy studies. In 
fact, it has become quite apparent that we can 
gain enough referrals of patients to support a 
clinical radiation therapy program. At the pres- 
ent time the principal need is to provide for 
additional beds for this Branch. 

This past year has also seen the creation of 
the Immunology Branch to meet the needs of 
the National Cancer Institute to further its re- 
search goals in the immunological aspects of 
cancer. 

The last items deal with the assignment of 
space in Building 14D to the National Cancer 
Institute for the support of a large animal sur- 
gery which will permit the development by the 
Surgery Branch of a large animal surgery pro- 
gram. 

These constitute specific items to which the 
Clinical Director desires to call attention, the 
reports of the Branch Chiefs should be consult- 
ed for specific details and accomplishments of 
each branch. 

Amino Acid Metabolism 

Studies of amino acid metabolism have con- 
tinued. These studies are designed to elucidate 
the physiologic and biochemical phenomena re- 
sponsible for the entry of amino acids into 
mammalian cells and to clarify the relation- 
ships between rates of amino acid transport 
and subsequent intracellular utilization and to 
define the significance of defects in amino acid 
transport in man. Initially this work was con- 
ducted in vitro using the rat kidney cortex slice 
as the tissue model. However, during the past 
year several other tissues have been studied 
and one of the surprising findings has been the 
distinct differences noted in amino acid trans- 
port mechanisms between different tissues 
both in man and the experimental animal. For 



NATIONAL CANCER INSTITUTE 



49 



instance, alpha aminoisobutyric acid, a model 
amino acid which is actively transported by the 
kidney, is not concentrated by intestinal mu- 
cosa despite the anatomic similarities in these 
two tissues. Secondly, in the cystinuric kidney 
no defect in cystine transport has been demon- 
strated whereas a marked defect in cystine up- 
take was shown in the intestine. Furthermore, 
human leukocytes from cystinuric patients 
showed no defect in transport of lysine or cys- 
tine in contrast to the results in kidney and in- 
testine. These and other findings suggest that 
there are important biochemical differences in 
cell membranes structure in different tissues, 
and that indeed, mechanisms of genetic control 
are also different in different tissues of the 
body. 

The role of the sodium ion in amino acid 
transport has been studied for the past two 
years. It has been shown in many tissues that 
removal of the sodium ion markedly interferes 
with mediated transport of a variety of amino 
acids. This sodium dependence has been studied 
in the intestine, kidney, leukocytes and rat 
bone. These studies indicate that the sodium 
ion may have more than one role in amino acid 
transport. It has been well shown that the 
transport of sodium across the cell membranes 
is an important regulator of cell respiration 
and hence, the coupling of transport mechan- 
isms to energy may well be regulated via so- 
dium transport. In addition, however, the so- 
dium ion has been shown to be necessary for 
transport systems in the absence of metabolic 
energy indicating a direct role in the passage 
of amino acids across the membrane region. 
Our studies have shown that the sodium ion 
stimulates influx of amino acid into the cell 
rather than affecting the efflux process and 
that the increase in movement of amino acid in 
the presence of the sodium ion is associated 
with an increase in net water movement across 
the intestinal wall. The phenomena of sodium 
dependence has also been used to study trans- 
port mechanisms for the neutral amino and 
imino acids in bone in the following way. Bone 
cells, or slices, incubated in the presence and 
absence of the sodium ion demonstrated that 
proline and hydroxyproline showed a complete 
dependence on the sodium ion for entry where- 
as glycine and alanine were only partially sen- 



sitive to this ion. Furthermore, when sodium 
ion was removed from the incubation medium, 
proline and hydroxyproline no longer inhibited 
glycine entry nor was the entry of proline 
affected by glycine or alanine. These studies 
show clearly that the amino and imino acids 
have different transport mechanisms and indi- 
cate that the sodium ion may indeed enter into 
linked flux relationships with amino acids and 
that these sodium-amino acid complexes in the 
cell membrane differ for different amino acids. 

We have continued to explore the nature of 
the transport defects in cystinuria, and have 
been led to the conclusion that cystinuria is in- 
deed two distinct diseases. This conclusion is 
based on the following evidence: first, two pa- 
tients with documented homozygous cystinuria 
showed normal transport of cystine and lysine 
by gut mucosa in contrast to the severe defect 
shown in twelve other cystinuric patients. 
These in vitro findings were corroborated by 
oral loading studies with cystine which indicat- 
ed that those cystinuric patients who could 
transport cystine in vitro were also capable of 
absorbing cystine from the gut while those sub- 
jects who failed to transport in vitro were un- 
able to absorb cystine to any significant degree. 
Additional evidence for genetic difference was 
made available through study of families of the 
cystinuric patients. These studies showed that 
the cystinuric subjects who were capable of 
transporting the dibasic amino acids and cys- 
tine by the intestine could be identified in the 
families by hyperexretion of lysine and cystine 
in the urine while no urinary abnormalities 
could be demonstrated in the families of those 
patients with the severe gut lesion. This leads 
to the conclusion that cystinuria must result 
from two separate autosomal defects with sim- 
ilar or identical urinary amino acid findings 
but with different manifestations regarding 
the intestinal tract and mode of genetic trans- 
mission. We have also investigated further the 
puzzling observation that cystine transport in 
the human cystinuric kidney in vitro was not 
defective. This led to the fundamental question 
of why these people excreted so much cystine. 
Renal arterio-venous differences were per- 
formed in two cystinuric patients and essen- 
tially no arterio-venous difference for cystine 
or cysteine were demonstrated in either case. 



50 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



These results are not compatible with a simple 
failure of tubular reabsorption of cystine by 
the kidney and suggest that the cystine excre- 
ted in large quantities in cystinuria may well be 
formed in the kidney by defective intrarenal 
metabolic processes. 

Calcium Metabolism 

During the past year the analysis of calcium 
kinetics in normal subjects and patients with 
metabolic bone disease has continued. In this 
work, calcium balance studies have been com- 
bined with Ca*' disappearance from the blood 
and appearance in urine and feces. In addition, 
Ca*^ has been monitored externally over the 
thigh and ankle. The data have been analyzed 
using a digital computer multicompartmental 
approach. The results in ten normal young men 
provide the first homogenous group of normal 
subjects studied this extensively. They indicate 
that in the period from 1 minute to 20 days 
after intravenous injection, Ca*^ exchanges 
with four body pools which do not have clear 
anatomic definitions. These studies provide 
quantitative estimations of the rate of transfer 
of calcium between these compartments as well 
as the rate of movement of stable calcium from 
the gut into the body, from the blood into the 
intestine, from the blood into the urine and 
from blood and tissue compartments into non- 
exchanging bone. The mathematical analysis 
has been programmed to yield numerical values 
for the uncertainties in the various parameters 
so that future studies of metabolic bone disease 
will have a suitable frame of reference. The re- 
sults obtained with this compartmental anal- 
ysis have been compared with those obtained 
using a simple one compartment model. This 
comparison has demonstrated that marked dis- 
crepancies result if only a single compartmen- 
tal model is used. Three patients with osteope- 
trosis or marble bone disease have been stud- 
ied. These patients show a markedly enlarged 
miscible calcium pool. However, even more 
striking has been the curves of appareance of 
calcium over specific bone sites in the osteope- 
trotic patients. These studies reveal a marked 
slowing of removal of calcium from the specific 
bone sites and analysis to date suggests that 
this slowing may be due to a specific decrease 



in the rate constant for movement of calcium 
from the most slowly exchanging calcium com- 
partment. These results would be compatible 
with the histologic findings suggesting the 
retention of calcified cartilage matrix as an im- 
portant feature in the pathogenesis of this dis- 
ease. Additional studies are in progress to de- 
termine whether age, sex or diet influence the 
measured parameters. 

Nucleic Acid and Pyrimidine Metabolism 

In continuation of the studies concerning the 
elevated urinary pseudouridine uric acid ratio 
found in chronic lympatic leukemia patients, 
investigations were performed on the ribonu- 
cleic acid formed by normal small lymphocytes 
and small lymphocytes from patients with 
chronic lymphocytic leukemia. It was found 
that under the incubation conditions employed, 
the rapidly labelled ribonucleic acid had the an- 
ticipated high sedimentation coeflficients of 35 
to 45 S and were in large percentage hybrid- 
izeable to human cell DNA. There was very 
slow conversion of only a small portion of this 
RNA to ribosomal nucleic acid unlike the 
events seen in dividing cells, such as KB cells. 
Very small amounts of S-RNA were synthe- 
sized. Thus, it is difficult to account for the ex- 
cess pseudouridine production on the basis of 
RNA production by the small lymphocytes. 

Further studies of lymphocyte metabolism 
indicated that these cells are producing signifi- 
cant amounts of major classes of histones even 
when they do not produce DNA. Investigations 
of histone fractionation by preparative acryl- 
amide electrophoresis are continuing. 

Negligible amounts of polyuridylic and pol- 
yadenylic have been found to occur naturally 
in E. coli when searched for by hybridization 
techniques 

Studies are continuing on the phenomenon 
observed in KB cells in which, under proper 
conditions azaguanine is incorporated solely 
into 4S transfer RNA. 

Nucleic acid hybridization studies of biologic 
phenomenon are continuing in several direc- 
tions. Applications of the methods to classifica- 
tion of mycoplasma have indicated that the 
method has great sensitivity in detecting strain 
differences among organisms and gives an in- 



NATIONAL CANCER INSTITUTE 



51 



dependent method of classifications of these or- 
ganisms that in most conditions agrees with 
the serologic classification. 

Efforts are continuing to detect viral DNA 
in viral induced tumors. Studies of RNA pro- 
duction in adenovirus infected cells indicate 
that viral messenger RNA is produced in the 
cells that are continuing to produce their own 
host-complementary RNA. In addition a com- 
ponent of RNA similar but chromatographi- 
cally distinct from transfer RNA appears in 
adenovirus infected KB cells. 

Studies of hemoglobin biosynthesis by nor- 
mal reticulocytes have indicated a lag of incor- 
poration of radioactivity into the alpha as com- 
pared to the beta chain or finished hemoglobin 
suggesting accumulation of precursor pool of 
alpha chain material. 

In thalassemia the situation is reversed in 
long term incubations and the alpha chain of 
finished hemoglobin has a higher specific activ- 
ity than the beta or gamma chains consistent 
with the hypothesis of alpha chain overproduc- 
tion in these patients. 

Studies of plasma protein synthesis in hepa- 
tectomized dogs and monkeys, have indicated 
small but quite significant amounts of synthe- 
sis of alpha and beta globulins in these ani- 
mals. 

The Metabolism of Plasma Proteins 

The technique for the direct measurement 
for the absolute synthetic rate of liver-made 
proteins using carbon^* labelled bicarbonate 
was compared to the indirect method of meas- 
uring protein synthetic rate using I'^^ la- 
belled proteins. The latter method is valid 
under steady state conditions. The rate of albu- 
min synthesis was estimated by both methods 
in six subjects without serum protein abnormal- 
ities and five patients with protein losing gas- 
troenteropathy. There was a very close agree- 
ment between the estimates of synthetic rates 
by both methods. It is therefore felt that the 
technique for the direct measurement of pro- 
tein synthetic rate developed will be of consid- 
erable value in the study of the factors con- 
trolling protein synthesis in man. 

The metabolism of albumin, gammaglobulin 
:IgG), B2A globulin (IgA) and gamma macro- 



globulin (IgM) was studied in a group of 10 
patients with hypogammaglobulinemia due to 
defective synthesis. This group of patients in- 
cluded subjects with congenital defects in 
gamma globulin synthesis, idiopathic acquired 
hypogammaglobulinemia and hypogammaglo- 
bulinemia secondary to thymomas or chronic 
lymphocytic leukemia. In each of the patients 
the primary disorder of protein metabolism 
was a defect in the synthesis of the three im- 
munoglobulins. Four of the patients had an as- 
sociated gastrointestinal disorder with secon- 
dary gastrointestinal protein loss and a short 
survival of each of the proteins. Each of the 
remaining patients had a normal survival of al- 
bumin, macroglobulin (IgM) and B2A globulin 
(IgA) but had a markedly prolonged survival 
of gammaglobulin (IgG). This finding is in ac- 
cord with the hypothesis that there is an indi- 
rect correlation between the survival half time 
of IgG and the serum concentration of this pro- 
tein. This finding appears to be best explained 
by the presence normally of a saturable system 
protecting IgG molecules from catabolism. As 
the concentration of IgG molecules decreases a 
larger fraction of the total number of IgG 
molecules are protected by this saturable sys- 
tem and the survival of IgG is prolonged. 

The metabolism of albumin and the three 
major classes of immunoglobulins was studied 
in a group of 18 patients with myotonia dystro- 
phica. In these patients the serum concentra- 
tion and rates of synthesis and catabolism were 
normal for albumin, IgA, and IgM. However, 
the majority of the patients had a significantly 
reduced concentration of IgG globulin. It was 
found that the catabolism of normal IgG was 
accelerated in these patients with half times 
averaging 12 days compared to over 24 days in 
17 normal control subjects. IgG obtained from 
patients with myotonia dystrophica had a simi- 
lar short survival in these patients but sur- 
vived normally in control subjects. The rate of 
synthesis of this protein, however, was normal. 
The reduced serum protein was thus entirely 
accounted for by an increased rate of break- 
down of IgG molecules. No such hypercatabol- 
ism of IgG was noted in approximately 20 
other patients with neuromuscular diseases. 
Thus patients with myotonia dystrophica ap- 



52 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



pear to have a unique immunoglobulin abnor- 
mality an isolated hypercatabolism of IgG. 

The metabolism of the immunoglobulins was 
also studied in patients with ataxia telangiecta- 
sia. This disease is characterized by progres- 
sive cerebellar ataxia, sinopulmonary infection, 
oculocutaneous telangiectasia, skin anergy, 
thymic abnormalities, and in a significant 
number (8 of 40) of cases associated reticu- 
loendothelial malignancies. The immunoglobu- 
lin levels in seven such patients were studied. 
The major abnormality was an almost com- 
plete absence of IgA globulin in six of the sub- 
jects and a reduced level of this protein in the 
remaining patient. Each of the patients had a 
normal survival of IgG, IgM and albumin. 
Each of the five patients studied had an ex- 
treme defect in the synthesis of IgA globulin. 
Four of these patients had in addition an ex- 
ceedingly short survival of IgA globulin indi- 
cating that combined defects in both IgA 
synthesis and catabolism were present. It was 
demonstrated that the patients with ataxia tel- 
angiectasia with an absence of IgA in the se- 
rum had significant quantities of IgA in their 
saliva. It was shown by immunofluorescent stud- 
ies that there were significant quantities of 
this protein in the salivary acinar cells. This 
finding is in accord with the observation of To- 
masi and co-workers that there are two related 
IgA type proteins, one formed in the plasma 
cells and the other formed in excretory tissues 
such as the salivary glands. It is presumed that 
in these patients there is a defect in the plasma 
cell but not of the salivary IgA globulin syn- 
thesis. The association of disorders of the 
thymus, the disorders of immunoglobulin metab- 
olism and the frequent occurrence of malig- 
nant tumors in these patients presents an in- 
teresting area for speculation on the role nor- 
mally played by the immunological system in 
the prevention of neoplastic disease. 

Studies previously initiated in patients with 
excessive gastrointestinal loss of protein were 
continued. Patients with gastrointestinal pro- 
tein loss were shown to have a markedly 
reduced level of albumin, IgG and IgA, a moder- 
ate reduction of transferrin, IgM ceruloplas- 
min and normal levels of fibrinogen. Using io- 
dine labelled proteins it was shown that there 
was a marked increase in the fractional catabol- 



ic rate for albumin, IgG, IgA, IgM and cerulo- 
plasmin in patients with gastrointestinal pro- 
tein loss. In general, the increase in the frac- 
tional catabolic rate over normal was quite 
comparable for the different proteins for a 
given patient, suggesting that there was bulk 
loss of plasma or of material with a compa- 
rable protein composition into the intestinal 
tract in these subjects. The synthetic rate for 
IgG, IgM was normal or slightly increased in 
each of the patients studied with the exception 
of one patient with regional enteritis who had 
high synthetic rate for IgC. This suggests that 
a reduced serum concentration of the immuno- 
globulins is not a potent stimulus for their 
synthesis and that other factors such as expo- 
sure to antigens are the major stimuli to im- 
munoglobulin production. 

A combined technique for the study of pa- 
tients with protein losing enteropathy was in- 
troduced using the simultaneous administra- 
tion of I"^ labelled albumin and chromium^^ 
labelled albumin. Using these combined studies 
the plasma volume, protein pool sizes, the rate 
of protein catabolism, the fraction of the intra- 
vascular pool lost into the intestinal tract daily, 
and in the steady state the synthetic rate can 
be determined. In normal subjects it was found 
that approximately 2 to 10% of the albumin 
catabolism could be explained by gastrointes- 
tinal protein loss. In patients with increased 
gastrointestinal protein loss, 60% of the circu- 
lating albumin pool could be lost into the gas- 
trointestinal tract each day. The major disad- 
vantage in the technique is that chromium" is 
gradually eluted from the serum protein. Be- 
cause of the short apparent survival of chro- 
mium'^ labelled albumin, copper" labelled 
ceruloplasmin has been developed as a tech- 
nique for quantitating gastrointestinal protein 
loss. This material was evaluated in studies in 
dogs, normal man and patients with gastroin- 
testinal protein loss. This material appears to 
fulfill better the major requirements for an 
ideal label for the quantitation of gastrointes- 
tinal protein loss than any previously proposed 
material. The radioactive copper is incorporat- 
ed into ceruloplasmin and becomes an integral 
part of the molecule throughout its life span 
without altering its metabolism or distribution. 
There is relatively little absorption of the ra- 



NATIONAL CANCER INSTITUTE 



53 



diolabel from the intestinal tract and there is a 
relatively low level of excretion of the label 
into the intestinal tract unbound to the serum 
protein. The survival of copper labelled cerulo- 
plasmin and I"^ ceruloplasmin were compa- 
rable in all groups studied. In normal dogs and 
man less than 20% of the overall metabolism 
of this protein could be explained by loss into 
the gastrointestinal tract. Thus loss of cerulo- 
plasmin into the intestinal tract plays only a 
minor role in the metabolism of this protein in 
normal subjects. In contrast in patients with 
gastrointestinal loss such loss of ceruloplasmin 
is a major factor in the metabolism of the pro- 
tein. 

During the past year gastrointestinal pro- 
tein loss has been demonstrated in patients 
with constrictive pericarditis, regional enter- 
itis, Whipples disease, sprue, celiac disease, 
carcinoid tumors, generalized amyloidosis asso- 
ciated with neoplastic disease, agammaglobu- 
linemia, and patients with gastrointestinal 
allergy as well as in new subjects with the syn- 
drome of intestinal lymphangiectasia. Further 
studies of patients with intestinal lymphan- 
giectasia have revealed multiple immunological 
defects in these patients. In addition to the 
short survival of the immunoglobulins these 
patients have marked lymphocytopenia, they 
have absence of tonsillar tissue and have skin 
anergy as well as the ability to retain skin 
grafts for long periods. 

Previous studies in man suggested that catab- 
olism and to a lesser extent urinary loss con- 
tributed to the metabolic fate of Bence-Jones 
proteins. In these studies, patients with normal 
renal function had a much higher catabolic 
rate for Bence-Jones proteins than those pa- 
tients with an elevated blood urea nitrogen. In 
the present study the role of the kidney in the 
catabolism of Bence-Jones proteins, isolated 
L-chains of IgG proteins and intact immuno- 
globulin was studied in mice. Bence-Jones 
proteins of the L-chains type from both human 
and mouse origin were catabolized at a much 
slower rate in nephrectomized then in ureter- 
severed or control mice. Similarly, the catabo- 
lism of the L-chain fraction IgG molecules was 
markedly reduced by nephrectomy. There was 
no difference in the rate of catabolism of whole 
IgG, IgM or IgA molecules between the ne- 



phrectomized and ureter-severed mice. Thus 
the indogenous metabolism of Bence-Jones pro- 
teins and of the L-chains moiety of IgG occurs 
primarily in the kidney. The kidney, however, 
does not appear to be a major site of catabo- 
lism for the intact immunoglobulins. 

Porphyrin Metabolism 

A new method has been perfected for the 
chemical determination of aminoketones in 
very small quantities to (a) study urinary ex- 
cretion and (b) measure tissue enzyme activity 
of ALA synthetase, aminoacetone synthetase, 
and threonine dehydrogenase. A quantitative 
method for the measurement of delta-amino- 
leyulinic acid synthetase. (ALA synthetase) in 
homogenates has been developed and applied to 
studies of porphyria and the factors controll- 
ing heme synthesis. Using these methods it has 
been shown that ALA synthetase in mamma- 
lian liver is 1) the rate controlling enzyme in 
heme biosynthesis, 2) an inducible mitochon- 
drial enzyme and 3) an enzyme whose induc- 
tion is inhibited by glucose. 

In a patient with acute intermittent porphy- 
ria, it has been shown that the increased excre- 
tion of porphyrin precursors is a result of a 
markedly increased hepatic level of ALA 
synthetase. This is the first example of an 
"overproduction disease" which has been local- 
ized to a specific enzyme. It is also the first ex- 
ample of the so-called "glucose effect" in man, 
i.e., the ability of glucose to inhibit the induction 
of an enzyme. 

A new pathologic entity has been discovered 
in a patient who expired from acute intermit- 
tent porphyria. This consists of a specific de- 
struction of the supraoptic and paraventricular 
nuclei. This is related to the syndrome of "in- 
appropriate secretion of antidiuretic hormone" 
in acute porphyria which was first reported by 
this laboratory several years ago. 

Studies of the factors involved in the control 
of heme synthesis and the relationships of this 
control to the level of heme protein enzymes 
have been conducted in mammalian liver. 
Tryptophane pyrrolase (TPO) is a heme con- 
taining enzyme which has served as a model 
system. Injection of allylisopropylacetamide 
(AIA) leads to induction of ALA synthetase 



54 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



which has a half life of 67-74 min. as deter- 
mined by inhibition of synthesis by puromycin. 
The increased production of heme resulting 
from induction of ALA synthetase leads to in- 
creased saturation of the apoenzyme of TPO by 
heme followed by an increase in the amount of 
total enzyme protein. Preliminary data suggest 
that the increased TPO protein level results 
from a longer half life of the protein which 
may result from stabilization of the protein by 
heme. The induction of ALA synthetase and 
the rise in TPO level which follow administra- 
tion of AIA can be prevented by actinomycin 
indicating enzyme induction is the mechanism 
of ALA synthetase increase. 

Administration of tryptophane causes an in- 
crease of the heme saturation (heme binding) 
of the TPO apoenzyme followed by induction of 
ALA synthetase. Only the induction of ALA 
synthetase is prevented by actinomycin. These 
data suggest that the degree of repression of 
hepatic ALA synthetase is related to the level 
of "free" or "available" heme in liver cells. The 
coordinated control of both heme and apoen- 
zyme levels can thus be maintained following 
changes in the protein moiety or in the heme 
synthetic pathway. Increased binding of heme 
by the apoenzyme diminished the repression of 
ALA synthetase, allowing for more heme pro- 
duction. Primary induction of ALA synthetase 
increases heme production which increases the 
amount of heme bound to the apoenzyme and 
prolongs its half life, thus raising its level. 
Preliminary data from other laboratories sug- 
gest that the tryptophane pyrrolase system 
may be a valid model for the study of the coor- 
dination of heme and globin synthesis in the 
marrow. 

Studies in adrenalectomized animals have 
demonstrated that adrenal steroids exert a per- 
missive effect on the induction of ALA synthe- 
tase in that injection of adrenal steroids causes 
no induction, but induction can not occur to a 
significant extent in their absence. 

The presence of a large variety of tumors in 
animals causes a decrease of hepatic ALA de- 
hydrase with progressive tumor growth. Poly- 
oma induced tumors appear to be an excep- 
tion to this phenomenon in that decreased 
hepatic ALA dehydrase is seen only occasion- 
ally with these tumors, regardless of tumor 



size or hematocrit. Repeated transplantation 
and the presence or absence of the polyoma 
virus do not affect the inability of polyoma in- 
duced tumors to lower the enzyme. Further 
studies are in progress to determine which fac- 
tors are related to the ability of a tumor to 
lower hepatic ALA dehydrase. 

Erythropoiesis 

Studies of the control of porphyrin biosyn- 
thesis continue. The initial studies of ineffec- 
tive erythropoiesis in man have been completed. 
Following administration of isotopically la- 
beled glycine in man, there are two peaks of 
the incorporation of isotope in the fecal bile 
pigments. The first peak reaches a maximum in 
a few days and the second a maximum in ap- 
proximately 120 days. The second peak repre- 
sents bile pigment produced from the catabo- 
lism of red cells at the end of the red cell life 
span. The origin of the first peak has not been 
clear. In the previously recorded studies in pa- 
tients it was evident that when erythropoiesis 
was absent by clinical and biochemical tests, 
that the amount of labeled glycine diverted to 
the early peak was markedly reduced to the 
order of less than 10% of that expected in the 
normal state. In order to study the early peak, 
bile fistula dogs were prepared and labeled gly- 
cine and labeled delta aminolevulinic acid 
administered. Previous data by Israels indicated 
that the early peak could be divided into two 
components. In order to study the early peak in 
the dog, both glycine-C" and ALA-C" were 
administered. With glycine-C", but not with 
ALA-C", in the normal dog there was evi- 
dence for a second component to the early 
peak. This component is markedly increased 
when erythropoiesis is increased as a result of 
bleeding and is markedly decreased when er- 
erythropoiesis is decreased by transfusion to 
polyeythemic levels. Analysis of the data from 
the normal polycythemic and the phleboto- 
mized dog indicates that in the normal approxi- 
mately two-thirds of the isotope appearing in 
the early peak is associated with erythro- 
poiesis; one-third is associated with another 
source. Studies by Schwartz at the University 
of Minnesota indicate that this source is prima- 
rily the liver and is due to the turnover of 



NATIONAL CANCER INSTITUTE 



55 



heme containing enzymes principally catalase 
in the liver. ALA-C^* was shown to be a very 
good precursor of labelled bilirubin and is now 
the method of choice for the preparation of 
carbon^* labelled bilirubin. A conversion of 
approximately 20% of the isotope to bilirubin 
has been achieved, compared to a yield of 
approximately one-tenth percent when glycine- 
2-C^* is utilized. 

Bilirubin Turnover 

A method has been developed for the meas- 
urement of bile pigment production by the use 
of the measurement of bilirubin turnover. Bili- 
rubin-C" has been prepared from bilirubin in 
the dog, has been crystallized, sterilized and 
brought into a physiological complex with albu- 
min and administered to patients. Samples of 
plasma were taken and the bilirubin separated 
into free and conjugated forms by the method 
of Weber and Schalm. The specific activity of 
the free bilirubin was then determined. The 
rate of change of specific activity of the plasma 
free bilirubin could be described in terms of 
two decreasing exponential components. Those 
data could be fitted to a simple three compart- 
ment model and the turnover of bile pigment 
calculated. In patients with normal erythroki- 
netics this ranged from 226 to 277 mgm/day. In 
patients with other evidence of increased bile 
pigment production by virtue of a decreased 
red cell life span, bile pigment production was 
increased to 600 mgm/day. These are the first 
measurements of total bile pigment production 
in man with a normal serum bilirubin, other 
than by a surgical drainage through a T-tube. 

An in vitro system for the measurement of 
hepatic glucuronyl transferase using bilirubin- 
C^* as a substitute has been developed. In the 
rodent a significant difference between male 
and female levels of enzyme activity has been 
noted. A small number of measurements have 
been made in man. 

In order to study the relationship between 
metabolic rate and erythropoiesis an indirect 
calorimeter has been constructed for the dog, 
in collaboration with the Metabolic Diseases 
Branch of the National Institute of Arthritis 
and Metabolic Diseases. This chamber provides 
for the measurement of carbon dioxide produc- 



tion and oxygen consumption. The chamber is 
now partially automated. It is anticipated that 
when fully automated continuous oxygen con- 
sumption and carbon dioxide production meas- 
urements can be obtained. It has been shown 
that it is possible to extrapolate the data from 
a 4 hour run to a 24 hour determination, thus 
shortening considerably the period required for 
the measurement of metabolic rate. In the nor- 
mal dog the metabolic rate average ranges 
from 40-50 kilocalories/kg/day. Preliminary 
studies in the dog given dinitrophenol indicate 
that an approximate doubling of the metabolic 
rate can be obtained but no change in total red 
cell volume was obtained after 84 days indicat- 
ing that at least in this animal oxygen con- 
sumption is not related to erythropoiesis when 
oxygen consumption was increased in this 
manner. In the dog made hypothyroid by ra- 
dioiodine, the metabolic rate is approximately 
one-half normal. In these animals there is a 
corresponding decrease in the total red cell vol- 
ume by half. Replacement therapy with thy- 
roid indicates that the metabolic rate and total 
red cell volume can be restored to the normal 
state. 

DERMATOLOGY BRANCH 

The clinical and laboratory research pro- 
gram of the Dermatology Branch continues to 
be primarily concerned with three major areas : 
(1) mycosis fungoides lymphoma; (2) epider- 
mal growth and differentiation; and (3) pteri- 
dine metabolism and pigmentation. 

Mycosis Fungoides Lymphoma 

The seemingtly fundamental relationship 
between immunologic reactivity of patients 
with this disease with the development and 
course of the disease prompts a continuation 
and intensification of efforts to explore such a 
relationship. The disease ordinarily begins 
with a reactive cutaneous dermatosis of eczem- 
atous type, which may persist for long pe- 
riods prior to the appearance of a disease proc- 
ess recognizable as a lymphoma. Some studies 
have been directed to patients in this early 
stage of development, such as evaluating their 
general immunologic reactivity. Although these 
patients are found to be within the range of 



56 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



normal in being capable of reacting immunolo- 
gically to various stimuli, the degree of reac- 
tivity compared to normals has not been 
established. Autologous transplantation and 
implantation of skin involved with early stages 
of disease has failed to show that the disease is 
transplantable at this stage. 

Several areas require extended exploration. 
Among these is the question of how to recog- 
nize malignant (neoplastic) lympho-reticular 
cells in an early stage infiltrate, composed of 
normal appearing cells for the most part. No 
morphological or behavioral characteristic is 
knovm that would help identify such cells. 
Early lesions injected with tritiated thymidine 
reveal that many cells, morphologically resem- 
bling both benign and malignant cells, synthe- 
size DNA and hence undergo mitotic division. 
The ability of cells from such an infiltrate to 
react in vitro to immunologic challenge hope- 
fully will provide essential data in this regard. 
The addition of an electron microscopic facility 
into the research program hopefully will yield 
important information on morphologic recogni- 
tion of early malignant cells. 

An encouraging, but yet unexplained phenom- 
enon, is the observation made in several pa- 
tients to date that dinitrochlorobenzene 
(DNCB) applied to early lesions of mycosis 
fungoides causes the lesion to clinically disap- 
pear. Whether this event is due to a hypersen- 
sitivity reaction or to a direct chemical effect 
has not yet been established, but studies to 
settle the question have been initiated. 

Epidermal Growth and Differentiation 

The governing relationship, other than a 
simple nutritive one, between epithelial cell 
populations normally and in neoplasia remains 
an area under active exploration. The fact that 
epithelial cells in tissue culture, under the 
highly artificial conditions of defined chemical 
media, readily depart from the normal both 
morphologically and functionally indicate that 
normalizing factors in the native environment 
is important in carcinogenesis, and perhaps in 
the course of the neoplastic process. Also, the 
fact that successful therapy of some types of 
epithelial tumors is not in itself cytocidal sug- 
gests that alterations of environmental condi- 



tions are operative in altering the growth and 
course of such tumors. The known therapeutic 
eflfect of topically applied 5-fluorouracil is be- 
ing examined for its possible mode of action on 
keratoses (pre-malignant lesions) and basal 
cell tumors. Specific alterations of the physical 
environment of some lesions are also being 
performed to assess how these modify tumors 
such as superficial basal cell epitheliomas and 
keratoacanthomas. 

Pteridine Metabolism and Pigmentation 

In the course of study on the mechanism 
whereby a known microorganism, utilized for 
the purpose, converts tyrosine and coumarin to 
a pigment, attention was directed to the hy- 
droxylating (oxidative) systems involved. 

The finding of two, and most likely three, dif- 
ferent hydroxylating systems in this organism 
currently under study offers a rather unique 
opportunity for studying the mechanism (s) of 
action of enzymatic hydroxylation. All three 
systems have characteristics in common and 
yet sufficient differences to make a comparative 
study quite revealing. It is hoped that study of 
the common cofactor requirement for a flavin 
(FAD) will reveal the series of events occur- 
ring on the enzyme surface during hydroxyla- 
tion. 

DIAGNOSTIC RESEARCH BRANCH 

Office of the Chief 

Very brief summations of the activities of 
the Chief of the Diagnostic Research Branch 
are included in the two so-called "projects" 
that follow. Each is a compilation of several ac- 
tivities which could, undoubtedly, be presented 
as numerous individual projects. For example, 
the first project reported on has three sections 
and involves active association with over nine 
universities as Project Officer, and the review 
of over 2400 cytological preparations (early 
bladder cancer diagnosis and diagnosis of tu- 
mor cells in the peripheral blood) , the develop- 
ment of a new instrument (CYDAC) , daily dis- 
cussion with one or more of the contractors, 
and attention to administrative details involved 
in contract work. 



NATIONAL CANCER INSTITUTE 



57 



The second so-called "project" involves collab- 
orative activities on a part time basis with 
over 12 investigators. Much of the work on 
LaC leukemia, although very necessary, has 
provided, unfortunately, essentially negative 
experience. The few positive leads have been de- 
scribed under "Major Findings." It can be spec- 
ulated that additional work could have been 
accomplished were more guinea pigs available. 
With current techniques, the cost in animals 
for transplantation alone has prevented the 
construction of protocols which could lead to 
anything but preliminary interpretation. 

Finally, not emphasized in the project 
reports are supportive activities of the Chief of 
the Diagnostic Research Branch. These in- 
cluded : 

(1) Function as Guest Editor for Acta Cyto- 
logica in the development and compilation of 
the two issues on the Symposium on Tumor 
Cells in the Peripheral Blood. (Acta Cytolog- 
ica, 9: No. 1 and No. 2, pp. 1-188, 2965 

(2) Histopathological diagnosis of all slides 
prepared from the experiment on the effect of 
diethylnitrosamine on guinea pigs and rabbits 
(a by-product of an experiment undertaken by 
Dr. Rapp), and the preparation of the final 
manuscript which appeared in the Journal of 
the National Cancer Institute, 34: 4, April 
1965. 

(3) The enzymatic analysis of leucine amino 
peptidase on the serum of monkeys given car- 
cinogens by Dr. Margaret K. O'Gara and Dr. 
Roger O'Gara, undertaken as a possible means 
of early diagnosis of liver carcinoma. An ab- 
stract was presented at the meeting of the Fed- 
eration of American Societies for Experimen- 
tal Biology in April by Dr. Roger O'Gara. 

Presumably, these latter two will have been 
reported elsewhere. 

ENDOCRINOLOGY BRANCH 

The Endocrinology Branch continues to 
represent a group of independently active in- 
vestigators who effectively pool their clinical 
and experimental skills for the advancement of 
their respective studies as well as for the main- 
tenance of a high standard of medical care. The 
blending of these skills provides a favorable 



climate for the development of the Branch's 
varied research efforts. 

Further extension of our therapeutic efforts 
in the field of trophoblastic disease now pro- 
vides a practical basis for the virtual eradica- 
tion of the advanced forms of choriocarcinoma 
initially brought under study. By the proper 
application of our current diagnostic and thera- 
peutic procedures to all pregnant women al- 
most all women can expect to avoid extensive 
trophoblastic disease without surgical inter- 
vention. Moreover, very few, if any, women 
should henceforth find it necessary to lose their 
reproductive function by hysterectomy in an 
attempt to eliminate early trophoblastic dis- 
ease. 

Our growing knowledge of the complexities 
of steroid hormone metabolism in normal and 
in pathological states is the product of increas- 
ing sophistication in analytical methodology, 
the main elements of which are the use of 
tracer methods and gas chromatography. This 
is a vivid example of how great an impetus is 
sometimes provided a field of investigation by 
the development of new tools. The enormous 
advances in our understanding that now attend 
the quantitatively reliable determination of 
hormone secretion rates and production rates 
as opposed to the old urinary excretion studies 
are evident from the reports from our steroid 
group. 

The molecular approach to disease analysis 
is complemented by the newer developments in 
chromosomal analysis. These innovations have 
illuminated many endocrinological syndromes 
and particularly those related to gonadal ab- 
normalities. The genetic relationships of these 
syndromes with each other and with other 
forms of hereditary abnormality of numerous 
somatic systems have become a matter of 
prime interest to the modern endocrinologist. 
Our group's firm orientation in the theory and 
methodology in this field represents a major 
contribution to our program. 

The endocrinologist must also now be an im- 
munologist of sorts. Our group has participat- 
ed effectively in the development of new and 
stimulating techniques for the immunoassay of 
the pituitary hormones as well as in a further 
characterization of these hormones as antigens. 
These observations provide a challenging op- 



58 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



portunity for the further exploration of these 
techniques in the study of normal and abnor- 
mal endocrine states. 

Extended observations make it increasingly 
clear that much that has been learned about re- 
productive and endocrine physiology in the ro- 
dent has little or no applicability to man or to 
the higher primates. Notwithstanding the 
great logistic burden imposed by work with 
monkeys, we have pursued an active study of 
menstrual function and endocrine aspects of 
pregnancy in the monkey. The dispensibility of 
the ovaries from early pregnancy in the mon- 
key is in direct contrast to the situation in the 
rat. Hence much of our ideas of therapy of ab- 
normal uterine function in women based on ro- 
dent studies will have to be revised. 

Neuro-endocrine studies are facilitated 
through the technique of ectopic transplanta- 
tion of endocrine organs. Our observations es- 
tablish that neuro-endocrine relationships are 
subject to experimental modification through 
prolonged alteration in the endocrine status of 
the animal as a whole. These data relate di- 
rectly to phenomena of aging with its atten- 
dant endocrine and oncologic changes. 

Administratively, our group appears to have 
evolved a modus operandi which is at once 
congenial and effectual. The operating prin- 
ciple is that of least and therefore, we hope, 
best government. The senior staff functions on 
an alternating basis in such a way as to pro- 
vide each member with maximum continuity of 
opportunity for sustained research effort. This 
in turn provides ample opportunity for the guid- 
ance and stimulation of the clinical and re- 
search efforts of the junior staff. The latter 's 
enthusiastic participation in the Branch's work 
is again reflected this year by an early request 
for a third year by one of our first year men. 
However, our second year group this year 
yielded no candidates for a third year, largely 
because of prior commitments elsewhere. 

Unfortunately, limitations of space and facil- 
ities preclude our acceptance of more Visiting 
Scientists notwithstanding increasingly numer- 
ous requests for such opportunities with our 
group. This may be regarded as a major defi- 
ciency in our program since we do profit so 
much from the stimulus provided by the pres- 



ence of such individuals during their stay among 
us. 

It may be anticipated that the coming year 
will provide a unique opportunity for the more 
complete exploration of many of the methodo- 
logical developments which have characterized 
this year's work. 

It is always a pleasure to indicate our warm 
appreciation for the sustained moral and ma- 
terial support extended the Endocrinology 
Branch by NCI. 

IMMUNOLOGY BRANCH 

The Immunology Branch was established less 
than a year ago from components of the Metab- 
olism Service and the Diagnostic Research 
Branch. It consists of 5 senior investiga- 
tors and three clinical associates, plus sup- 
porting personnel. In the past year, new labo- 
ratories have been outfitted in Building 10, and 
the Immunochemistry Section moved from the 
Auburn Building in Bethesda to Building 10 in 
January, 1965. 

Research in the past year has been concerned 
with molecular and biologic aspects of immu- 
nity and largely has been an extension of pre- 
vious interests of the principal investigators. 
New lines of work with antigens of carcinogen 
induced tumors in animals and cellular (leuko- 
cyte) antigens in man have begun and show 
considerable promise for fruitful development. 

A notable success was the discovery of a new 
class of human immunoglobulin, termed im- 
munoglobulin D, IgD, or yd, by Dr. David 
Rowe, a guest worker from Birmingham, Eng- 
land, and Dr. Fahey. This is the first new 
class of immunoglobulins discovered in man 
since 1956, and developed directly from studies 
of the abnormal proteins that occur in multiple 
myeloma. With the discovery of IgD, the total 
number of immunoglobulin classes recognized 
in man are four, these components being IgG, 
IgA, IgM and IgD (Ig = immunoglobulin) . 

The new component, IgD, is present in se- 
rum of most normal subjects and constitutes 
less than 1% of the total immunoglobulins. 
This component can be formed in malignant 
plasma cells, and at least five cases of multiple 
myeloma producing abnormal D-myeloma pro- 
teins have been identified. The role of the IgD 



NATIONAL CANCER INSTITUTE 



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immunoglobulin in immunity remains to be de- 
termined. Immunological studies and studies of 
the changes of this immunoglobulin in disease 
are under investigation at the present time. 
The possible linkage of the structural gene for 
the 8 polypeptide chain, the characteristic 
chain of IgD, to any of the genes controlling- 
other immunoglobulins also remains to be de- 
termined. 

Studies of human immunoglobulins were ex- 
tended by the identification of four subclasses 
of the IgG immunoglobulins (7S-y2-glodulins) by 
Dr. W. Terry. These components, tentatively 
termed y2a, y2b, y2c and y2d, are present in 
normal serum as well as among G-myeloma 
proteins. These subclasses probably differ func- 
tionally, and Dr. Terry has shown that there 
are differences in skin-binding activity between 
these four classes of immunoglobulins. He is ac- 
tively extending this work now and has prelim- 
inary data indicating differences in the genes 
controlling these subclasses of immunoglobulin. 

Investigation in mice also revealed a new 
class of immunoglobulins and new subclasses 
of IgG or 7Sy-globulin. Five immunoglobulin 
forms are now recognized in the mouse: IgM, 
IgA, 7Syi-, TSyoa- and 7Sy2b-globulin. The 7S im- 
munoglobulins were found to differ in genetic 
control and in functional activity in terms of 
the skin-sensitizing ability in homologous and 
heterologous species, and in response to im- 
munization. 

These findings in man and mouse indicate 
an extensive heterogeneity within the immuno- 
globulin family, but also indicate an order to 
the levels of heterogeneity. Appreciation of the 
various levels of heterogeneity will facilitate 
correlations between structural genes and spe- 
cific polypeptide chains and between structure 
and function in the immunoglobulin molecules. 
Efforts will be expended in the coming year to 
identify specific amino acid sequences, carbohy- 
drate content and other features of the immun- 
globulin molecule which are characteristic of 
specific antibodies and of molecules with partic- 
ular genetic factors and immunologic func- 
tions, such as complement fixation, skin fixa- 
tion or transfer across the neonatal intestinal 
tract, etc. 

Studies in various conditions of immuniza- 
tion in mice revealed that specific immunoglob- 



ulins were affected differently depending on; 
the mode of administration and the presence or 
absence of adjuvant. This has profound biolog- 
ic effects, because not all antibodies have the 
same functional characteristics. There also 
seem to be genetic differences, which indicate 
that genetic factors control immune response. 

The possibility of genes controlling immune 
response was investigated in man by extension 
of studies in a patient who has selective im- 
munoglobulin deficiency. This patient has a 
very profound deficiency of IgG and IgA im- 
munoglobulins, but has a superabundance of 
IgM and IgD. An extensive series of immuno- 
logic, physiochemical, cytologic, biosynthetic, 
immunofluorescent, electron microscopic and 
metabolic studies indicated that this patient 
was able to produce the particular polypeptide 
chains unique for IgG and IgA only in very 
small amounts. A defect in the regulatory proc- 
ess, very likely in a regulatory gene(s) con- 
trolling the synthesis of these two polypeptide 
chains (and, hence, of the IgG and IgD forms 
of immunoglobulin molecules), is the probable 
basis for this abnormality. Further studies of 
this phenomena are indicated with patients 
who have other forms of immunoglobulin dis- 
orders, because investigation of the regulatory 
defects will probably provide considerable in- 
sight into the exact mechanisms by which im- 
munoglobulins are formed. 

The mechanisms of immunoglobulin forma- 
tion are just beginning to be investigated. It is 
hoped that in vitro studies with isolated sys- 
tems as well as appropriate in vivo investiga- 
tions will illuminate the processes by which an- 
tigen recognition occurs, antibody response is 
initiated and immunoglobulin formation is ac- 
celerated in the normal state, as well as in dis- 
ease. 

Immunoglobulins, once S3aithesized, depend 
upon their physical properties to determine 
their length of survival. The macroglobulins 
(IgM) for example, are catabolized at four or 
five times the rate of the 7S IgG antibodies. Re- 
cent studies conducted with Dr. Sell have 
shown that even the IgG molecules are hetero- 
geneous in their catabolic properties. These 
studies were carried out in mice and showed 
that three subclasses of IgG differ in their cat- 
abolic properties, and their half-times may 



60 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



differ by as much as a factor of two. This is an 
important factor in determining survival of 
antibodies once formed, and strongly influences 
serum level of individual antibodies. Mouse 
macro- and mouse IgA globulins apparently 
have no homeostatic regulatory mechanism to 
help control the level of those molecules. The 
catabolism of these molecules, however, is 
much faster than of the IgG group. 

Catabolic studies will be extended to the IgD 
molecules and to the subclasses of human IgG 
immunoglobulins in the coming year. It seems 
probable that the normal human IgG immuno- 
globulins differ in their catabolic properties be- 
cause heterogeneity has been observed in the 
catabolism of normal IgG in previous studies in 
our own and other clinics. 

Immunochemical technics for the diagnosis 
of multiple myeloma and macroglobulinemia 
were extended to making available reagents for 
discerning Type I and Type II immunoglobu- 
lins. These are differences from the IgG, IgA, 
IgM and IgD classes noted above, and are 
based on differences in the light (L) polypep- 
tide chain properties. Reagents were supplied 
to Dr. Bergsagel, who has found that patients 
with Type I Bence Jones protein respond much 
better to chemotherapy with melphelan than 
patients with Type II Bence Jones proteins. 
This initial observation will have to be ex- 
tended and checked by other groups conducting 
chemotherapy studies. It also raises the ques- 
tion of whether the different forms of heavy 
chain noted above in the subclasses of IgG will 
make a difference in clinical course or the re- 
sponse to therapy of tumors forming these 
types of protein. 

Conduct of such studies will require more 
reagents and greater facilities than are avail- 
able within the Immunology Branch of the 
NCI. Because of this need arrangements have 
been made to have some of this work carried 
out by a suitable contractor. It is hoped, by 
means of this contract, to extend the facilities 
which are now available in only a few research 
laboratories to all of the clinics participating in 
the Cancer Chemotherapy Cooperative Studies 
of multiple myeloma and related diseases. Par- 
ticular emphasis will be upon a classification of 
the abnormal proteins and the relationship be- 
tween the type of abnormal protein and the 



prognosis and response to therapy and other 
clinical features of malignant disease. The 
diagnostic facilities also will be extremely val- 
uable in recognition of macroglobulinemic- 
lymphoma, and also should assist in the recog- 
nition of agammaglobulinemia and other im- 
munoglobulin deficiency syndromes. 

Clinical investigations in Waldenstrom's 
macroglobulinemia were continued with con- 
firmation of the value of plasmapheresis in re- 
ducing hyperviscosity symptomatology. It is 
anticipated that future work will be concerned 
with the distinctive cells occurring in chronic 
lymphocytic leugemia, Hodgkin's Disease, 
macroglobulinemic lymphoma, plasmacytomas 
and related malignant diseases. In order to 
carry out these studies, facilities have been 
established within the Immunology Branch, as 
well as in cooperation with the Medicine Branch, 
for characterizing the cellular antigens of leu- 
kocytes, with particular emphasis on the lym- 
phocytes and plasma cells. Studies have been 
initiated, both in mice and in man, for detect- 
ing the known cell antigens, as well as pro- 
cedures for identifying new antigens, which 
can be related to the malignant process or to 
the normal functional features of these various 
forms of cells. 

The Immunochemistry Section of the Im- 
munology Branch has been conducting studies 
on the antigenicity of carcinogen-induced tu- 
mors in guinea pigs and rabbits. Quantitative 
study of the antigens of hepatomas induced by 
nitrosamine, compared with liver from the 
same animal prior to treatment, as well as with 
normal liver obtained at the time the tumor 
was removed and liver from other animals of 
the same strain has been an important aspect 
of this work by Dr. Rapp and associates. Much 
more Forssman antigen was found in the tu- 
mor than in normal tissue. Studies are being 
extended to other antigen detecting systems to 
see if the differences are wholly quantitative 
between malignant cells, or whether there are 
qualitative differences as well. 

A new test for antigen-antibody reactions, 
which has great sensitivity as well as precision, 
has been developed by Drs. Borsos and Rapp. 
This test is termed the da-fixation and 
transfer test. By using their knowledge of the 
procedures of the mechanisms of complement 



NATIONAL CANCER INSTITUTE 



61 



activation, these investigators developed the 
new test, which should be exceedingly useful in 
studies of cytotoxic reactions in vitro as well as 
in the nature of the antibodies and cellular an- 
tigens required in this process. This is a very 
active program which should be extremely use- 
ful to many of the investigators in the NCI in- 
volved with the immunology of neoplasms. 

Clinical studies will develop in the direction 
of efforts to find immunochemical uniqueness 
of neoplastic cells, as well as the specific effect 
of malignant disease on the immune process. 
We hope to make some further investigations 
into the suppression of antibody formation 
that occurs in chronic lymphocytic leukemia, as 
well as the suppression of delayed hypersensitiv- 
ity that occurs in Hodgkin's disease, by means 
of studying isolated cells or tissues from pa- 
tients with these diseases. 

Studies of transplantation immunity in the 
Immunology Branch will be concerned with 
transfer of lymphocytes and other immune ac- 
tive cells. Collaboration with other Branches 
concerned with transplantation of other cell 
lines or organ systems is being developed. We 
now have a capacity for detecting the genetic 
determinants of human immunoglobulins, i.e. 
the Gm and Inv factors. This capacity should 
help in selection of donor-recipient transplan- 
tation combinations, as well as in efforts to de- 
termine the success of immune cell transfer. 
The systems for detecting cell antigens now 
under development also will be helpful in this 
work. 

Efforts will be made to selectively suppress 
immune response so that cell and tissue trans- 
plantation may take place without the hazards 
of bacterial infection, etc. that result from gen- 
eral suppression of immunity. 

Finally, efforts will be directed toward 
preparation of cell fractions with immunologic 
information. Some of these are expected to 
make antigens more potent and, hopefully, 
others may permit sensitization against specific 
foreign antigens. 

RADIATION BRANCH 

During the past year, the clinical and experi- 
mental research activities of the Radiation 
Branch have been redeveloped to complement 



its continuing service functions. This report 
will review the programs which have been un- 
dertaken and will describe the objectives of 
projects being established. 

A major effort was the comprehensive ap- 
praisal and reorganization of all radiothera- 
peutic procedures as a prelude to the initiation 
of new clinical research studies. During this 
period, treatment techniques have been stan- 
dardized, dosimetry planning procedures have 
been established for reproducibility from pa- 
tient to patient, and routines have been insti- 
tuted for the undertaking of clinical investiga- 
tions. 

Following this reorientation toward an in- 
creased emphasis on clinical research, a 
number of studies were undertaken. Primary 
in terms of patient accession numbers has been 
the evaluation of radiotherapy in Hodgkin's dis- 
ease. The value of intensive radiotherapy in 
localized Hodgkin's disease has been generally 
accepted, and the Radiation Branch has under- 
taken studies to investigate the role of radical 
radiotherapy in advanced Hodgkin's disease as 
well as the evaluation of extended radiotherapy 
in localized cases. Another area of intense in- 
terest is the use of total body irradiation in the 
chronic leukemias and disseminated lym- 
phomas. Although the number of patients ad- 
mitted to the total body irradiation studies is 
as yet small, some very promising results have 
been obtained in terms of clinical remissions. 

A pilot study combining radiotherapy and 
chemotherapy has been undertaken on Ewing's 
sarcoma. Two patients have received treatment 
and both are clinically well for periods exceed- 
ing the median duration of remission for this 
disease. Preliminary studies evaluating in- 
creased oxygen tension at atmospheric pres- 
sure for potential radiation enhancement have 
commenced as have controlled clinical radiobio- 
logical investigations of dose-time relation- 
ships. 

A cooperative study on endometrial carci- 
noma has been organized by Dr. John Marshall 
of the Surgery Branch, NCI. The Radiation 
Branch will serve as the radiotherapy center 
for this study, and will review the radiother- 
apy of all patients entered into the study by the 
participating institutions. The Acting Chief of 
the Radiation Branch serves as Chairman of 



62 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the Radiotherapy Subcommittee for the Coop- 
erative Study Group. 

A scanning device for the measurement of 
the transmission thickness of patients has been 
developed by the Physics Section of the Radia- 
tion Branch. The clinical application of this unit 
is underway and shov^^s considerable promise 
of increasing the accuracy of dosimetric calcu- 
lations rendered difficult by body density inho- 
mogeneities. 

Several laboratory research projects related 
to the chemotherapeutic and radiotherapeutic 
effects Qn mammalian tumor cells have been 
completed. The L1210 murine leukemia system 
has been employed and biological and cytoge- 
netic alterations induced in tumor cell popula- 
tions as a consequence of treatment have been 
investigated. Alkylating and antimetabolite 
drugs as well as ionizing radiations have been 
found capable of altering the growth kinetics 
of L1210 leukemia. The effect of these changes 
on sensitivity to treatment and attempts to cor- 
relate these findings with clinical observations 
are currently receiving attention. Other experi- 
mental studies using total body irradiation are 
designed to pursue a possible relationship be- 
tween tumor response and dosage-fractionation 
schedule in order to establish a model approach 
as the basis for systematic clinical trials of this 
therapeutic modality. 

A number of studies in collaboration with 
other areas have proceeded during the year. 
These include an evaluation of pre-operative 
radiotherapy in carcinomas of the upper air 
passages (and in other selected instances) with 
the Surgery Branch, NCI, the investigation of 
combined chemotherapy and radiotherapy in 
Hodgkin's disease with the Medicine Branch, 
NCI, and the continuing assessment of pitui- 
tary irradiation for acromegaly with the Na- 
tional Institute of Arthritis and Metabolic Dis- 
eases. The considerable clinical and non-clini- 
cal irradiation services, including consultative 
services, have been a continuing responsibility 
of the Radiation Branch. 

The expanding program of research has 
brought to the surface the acute need for the 
renovation of the physical facilities for both 
experimental and clinical purposes. The exces- 
sive amount of time required by professional 
staff for maintenance, combined with the rela- 



tive inflexibility of the present machines, are 
limitations to the research productivity of the 
Branch. The replacement of the outdated 
equipment with modern, flexible units is under 
consideration at the present time. 

SURGERY BRANCH 

The Surgery Branch of the National Cancer 
Institute continues to act in a dual capacity, 
that of carrying out its own specific clinical 
and laboratory investigations and that of pro- 
viding consultation surgical service to other 
branches of the Cancer Institute and to the 
clinical units of each of the other institutes of 
the National Institutes of Health. This service 
includes not only general surgical assistance, 
but urological, otolaryngological, gynecological 
and a portion of the thoracic consultations ne- 
cessitated by the broad scope of investigative 
clinic studies being carried out by the various 
institutes. 

There were 931 individual consultation re- 
quests answered by the Surgery staff, 306 on 
NCI patients, and 625 on patients from other 
institutes. 471 operative procedures were re- 
quired in carrying out these consultations, 215 
on NCI patients and 256 on patients from 
other institutes, 27% of which entailed a major 
surgical procedure. In addition, 356 surgical 
procedures were carried out on patients ad- 
mitted to the Surgical Branch, 58% of which 
were major operations. A total then of 895 sur- 
gical procedures have been performed during 
the 12 months covered by this report. The en- 
tire surgical consultation service was augment- 
ed by consultants from the community who 
saw 247 patients, 233 of which were non-malig- 
nant otolaryngolical problems. These consul- 
tants performed or assisted in 21 surgical 
procedures. 

The surgical Branch has not tried to direct 
its clinical interests in any one anatomical field, 
but has studied patients as they have been re- 
ferred to the branch. A rather aggressive surgi- 
cal approach to cancer has been carried out, in 
great part dictated by the fact that over 70% 
of the patients undergoing cancer surgery have 
been treatment failures before their referral 
and admission to N.I.H. 



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Malignant disease of the head and neck 
areas continues to be an anatomical site of 
prime interest to the Surgery Branch. The 
double blind preoperative radiation study of 
head and neck cancer patients now include 47 
patients. One thousand roentgens given to the 
tumor area 24 hours preoperatively has caused 
some increase in morbidity, but the followup 
time is too short to determine the effect the ra- 
diation may have on the end results. The study 
calls for untreated patients; for this reason 
numbers are accumulated very slowly. 

Survival by conventional means of therapy 
from paranasal sinus cancer is very poor due 
to tumor extension into the ethmoid, sphenoid 
and ptygeroid areas, resulting in local recur- 
rence in spite of the usual surgical or radiologi- 
cal techniques of treatment. 31 patients have 
now had a combined intracranial, transfacial 
en bloc resection of the paranasal sinus area, 
including the cribriform plate and medial orbi- 
tal walls, usually leaving the orbital contents 
intact. Preliminary followup suggests that 
with no increase in operative mortality, this 
aggressive procedure is resulting in an in- 
creased number of patients being given a 
chance of cure. An increased two year survival 
is now evident. 

Admission surveys of patients admitted with 
oral, pharyngeal and laryngeal cancer con- 
tinues to show a significant correlation between 
smoking and alcoholic intake and the anatom- 
ical site of their tumor and of the incidence 
of multiple primary head and neck malig- 
nancies. The morbidity and mortality re- 
lated to the surgical exploration of these ad- 
vanced cancers has been significantly lessened 
by the increased attention that has been direct- 
ed toward better selection of skin incisions, the 
use of antibiotic prophylaxis and the use of the 
cervical esophagostomy as a convenient and 
well-tolerated mode for prolonged tube feeding 
and keeping food away from healing suture 
lines. Of 16 patients who have had 19 sponta- 
neous ruptures of the carotid artery during 
postoperative convalescence over the past 10 
years, only 4 such occurrences have developed 
since 1961. This has in great part been due to 
the attention which has been directed to the 
above considerations. Alertness on the part of 
the hospital staff has provided control for 17 of 



these hemorrhages. 16 have been restored 
without complication to their pre-rupture 
status. 

Patients with carcinoma of the cervix con- 
tinue to be referred, having had previous defin- 
itive surgery, supracervical hysterectomy, in- 
adequate irradiation, and histories of never 
having had a Papanicolaou smear taken. We 
are convinced that this disease, which now 
ranks second in prevalence of cancers in wom- 
en, could in great part be controlled by either 
radiation or surgery if the disease was recog- 
nized in its earlier stages. This definitely can 
be done by the routine Papanicolaou smear 
technique. Our advanced stage of disease pa- 
tients, with carcinoma of the cervix, show an 
over all 43% five-year survival following ex- 
tensive surgery for advanced, usually recur- 
rent disease and an operative mortality of less 
than 10%. The patients who underwent radical 
hysterectomy have a 70% survival, anterior 
pelvic exenteration, 34 % ; and total pelvic exen- 
teration, 24%. 

As we are developing better techniques of se- 
lection of patients with advanced pelvic disease 
by aggressive surgery, the survival following 
radical surgery continues to improve. Unilater- 
al leg edema, sciatic distribution of pain, bilat- 
eral renal involvement, lymphangiography, 
renal and surgical physiological studies, all are 
clinical and diagnostic aids in determining fea- 
sibility of cure. For advanced tumors of the 
pelvic organs; vagina, cervix, uterus, bladder 
and rectum, surgery continues to offer the best 
chance of cure but this is not done without, at 
times, severe morbidity. Improved techniques 
of blood volume determinations, red cell survi- 
val studies, bowel sterilization techniques, re- 
nal and surgical physiological studies through 
isotope renography, vital sign monitoring and 
accurate fluid and chemical replacement deter- 
minations all combine to make exenterative 
surgery a practical and potent means of bring- 
ing under control advanced pelvic malignant 
disease. 

A study on preoperative bowel sterilization 
to determine the efficacy of a standard 3 day 
bowel sterilization previous to intestinal sur- 
gery has confirmed that 5 days of preparation 
is not necessary. Equivalent doses of Kanamy- 
cin and Paromomycin have been satisfactory in 



64 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



reducing postoperative complications and in 70 
patients, no instance of staphylococcal entero- 
colitis has been encountered. 

Lymphangiography, while an aid in preoper- 
ative studies, finds its greatest value in a guide 
for more complete lymph node dissection at the 
time of surgery. This procedure remains a rou- 
tine in the preoperative evaluation of all ad- 
missions with pelvic disease. 

The use of the ileal conduit as a mode of 
urinary diversion continues to be the most 
satisfactory means of diverting the urinary 
stream following bladder removal. This tech- 
nique of diversion both in short and long term 
studies results in few complications and pre- 
serves excellent renal function in nearly all in- 
stances, if attention is directed toward antibac- 
terial prophylaxis in preventing infectious 
complications. Of interest has been a review of 
13 patients who have undergone urinary diver- 
sion in spite of preoperative and confirmed 
operative evidence of one ureter being com- 
pletely blocked by tumor. Diverting this ureter, 
after high transection, into an ileal conduit, 
has resulted in all but 1 of the kidneys return- 
ing to function, half very satisfactorily and the 
others with a variable degree of impairment. 
Plastic reconstruction of a vagina in selected 
cases, intensive colostomy and urinary ileos- 
tomy instruction and guidance in patients who 
have had radical pelvic surgery can and does 
result in a normal, pleasant and productive 
life. 

During the past year, 9 patients with malig- 
nant melanoma have undergone 17 major sur- 
gical procedures in an attempt to eradicate 
their disease by wide local resection of the pri- 
mary tumor and incontinuity lymph node 
drainage area resection. An analysis of 35 pa- 
tients operated on more than 5 years ago for 
locally recurrent melanoma reveals only 8 still 
alive and free of recognized disease. 13 sur- 
vived more than 5 years while only 4 survived 
more than 10 years. It has become apparent 
that lesions of the scalp and face are prone to 
develop lymph node metastases in the parotid 
area and superficial parotidectomy is manda- 
tory if node dissection is to be carried out. It is 
also apparent that our survival results are 
most discouraging and with this in mind, sev- 
eral avenues of study are underway both in the 



experimental animal laboratory and in the clin- 
ic, such as intralymphatic administration of 
therapeutic radioisotopes or combined chemo- 
therapy infusions or perfusions, with potential 
techniques of bone marrow protection. 

An increasing number of extremity sar- 
comas, all recurrent following previous treat- 
ment, are being seen by the Surgery Branch. 
Selected instances of amputation and particu- 
larly hemipelvectomy, which allows removal of 
all extremity muscular and fascia, has been 
performed with no mortality and good physical 
rehabilitation. 

A long term survey of infectious complica- 
tions following cancer surgery shows that the 
patient who carries a pathogenic organism 
with him into the operating room, whether it 
be from the skin, nose or throat or particularly 
within the tumor, runs a significantly in- 
creased risk of developing an infectious compli- 
cation postoperatively. Staphylococcal infec- 
tions predominate but are more easily con- 
trolled and cause less patient morbidity than 
other usual hospital organisms. Antibiotic 
therapy remains a routine with all patients un- 
dergoing cancer surgery, Chloramphenicol be- 
ing the most satisfactory and frequently used 
drug. This very potent antibiotic has been ex- 
tensively used for five years by the Surgery 
Branch, without evidence of hematological 
complications. Bj^ employed high doses for a 
limited ten day interval, infectious complica- 
tions over the past year have been 7% as com 
pared to 22-30% with no antibiotic therapy. 

A combined laboratory and clinical program 
is underway in an effort to recognize, explain 
and quantitate a few of the many physiological 
parameters which change following extensive 
cancer surgery. Accurate and precise methods 
of measuring the total red cell volume, plasma 
volume, and extracellular fluid volume have 
thus far been developed and are currently in 
use. In addition, the neuroendocrine mecha- 
nisms controlling aldosterone secretion are be- 
ing studied and an attempt to develop an im- 
muno-assay procedure for the determination of 
circulating anti-diuretic hormone is being 
made. 

In a search for the culpable circulatory de- 
fect in refractory shock, all the usual hemody- 
namic and biochemical parameters thought to be 



NATIONAL CANCER INSTITUTE 



65 



important in shock are being studied, plus sev- 
eral methods previously unused in this setting. 
These latter include radioisotope clearance 
methods for measuring capillary blood flow, 
electrocapacitance plethysmography and indi- 
cator dilution methods for measuring regional 
blood flow and distribution of cardiac output 
and dextran molecular weight dispersion meas- 
urements as indicators of altered capillary 
permeability. The use of experimental animal 
models in the LD50 range and serial studies on 
patients undergoing extensive radical surgery 
should provide better information about the 
early changes developing in those instances 
where refractory shock eventually occurs as 
compared with the subjects in which refrac- 
tory shock does not develop. 

During the past year, little attention has 
been directed toward further refinements in 
techniques of isolating tumor cells from the 
blood of cancer patients. It remains apparent 
that if enough blood is sampled from the 
cancer-bearing patient, tumor cells will even- 
tually be isolated. Surprisingly enough little 
correlation has been noted between patient sur- 
vival and the presence or absence of circulating 
tumor cells. Tumor cell isolation, identification 
and interpretation of significance, from wound 
washings and drainages has similarly been 
unrewarding in most respects. Possibly due to 
better techniques of filtering and certainly due 
to better criteria of identification of individual 
cells by the cytologists, the incidence of tumor 
cells identification has decreased to such a low 
percentage cure as compared to previous years, 
that validity of the over-all study is questioned. 
We are, however, using proflavine hemisulfate, 
an acridine dye, as a tumoricidal wound wash- 
ing agent in a continuing clinical study. 

A comprehensive study of the human thora- 
cic duct lymph has been undertaken by the Sur- 
gery Branch. Suppression of the human im- 
mune response has been demonstrated with 
drainage of lymph and further studies are in 
process in an attempt to elucidate the active 
substance in lymph which inhibits man's abil- 
ity to form antibodies when challenged by a 
primary antigen and prevents normal rejection 
of skin homografts. Lymphocyte depletion has 
not been the answer in that immune suppres- 
sion does not occur when cell-free lymph is 



returned to the patient. Studies are in progress 
to determine the importance of serum immune 
globulins in this phenomenon. Recirculation of 
lymphocytes from the blood through lymphatic 
filters and then through the thoracic duct back 
into the systemic blood is being studied by 
means of radioactive tags on the cells and by 
chromosome markers. Storage of lymphocj^es 
is also being studied by means of vital stains 
and cell transformation in an attempt to learn 
more about the life span of human lympho- 
cytes. Production of immune globulins by tho- 
racic duct lymphocytes, in contrast with serum 
lymphocytes, is also underway. This method of 
human immune suppression has significance in 
organ transplantation since it has been shown 
to be well tolerated by man. Further investiga- 
tive work both in animals and in humans, is 
underway to determine the value of this method 
of immunologic suppression. 

Lesions of the renal vasculature causing hy- 
pertension have been of major interest to the 
Surgery Branch with studies directed toward 
improved diagnosis and surgical management. 
The isotope renogram is used as a screening 
test for renal disease, and is valuable for fol- 
lowing changes in renal function after revascu- 
larization procedures. Differential function 
studies supported by renal arteriography re- 
main the basic diagnostic procedures. After the 
Stamey test (during which urinary oxygen ten- 
sion is monitored), a cardiac phonocatheter is 
passed transureterally to each renal pelvis 
where sonic vibrations of the renal artery 
pulse are recorded. These ancillary diagnostic 
procedures provide new evidence regarding the 
renal hemodynamic and metabolic changes 
during ischemia. 

Laser energy has been established as an on- 
colytic agent for a wide variety of experimen- 
tal tumor systems. A technique has been devel- 
oped which permits the quantitative destruction 
of malignant tissue by laser energy. The de- 
structive capability of laser energy is depen- 
dent on the particular wavelength absorption of 
a given tumor. A technique to correlate the 
wavelength absorption properties of experimen- 
tal animal tumors and human tumors has been 
assessed. Pulsed laser energy can be used to de- 
stroy eflfectively, rapidly, precisely, and safely 



66 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



multiple malignant tumor implants. Very high 
energy laser impacts to the intact canine head 
have produced only superficial localized burns 
with no untoward effects. It appears that laser 
energy may be a potential adjunct to cancer 
therapy. 

The cooperative, prospective study of the 
primary therapy of endometrial carcinoma 
should provide an answer to an old problem. 
The different types of therapy under study are 
surgery alone, preoperative radiation and sur- 
gery, and surgery and postoperative radiation. 
Patients will be assigned to therapy at random. 
Utilizing the Surgery Branch, as the Study 
Center, data from cooperating institutions will 
be collected and analyzed. The study is planned 
to include 400 patients. Ultimate analysis of 
the data after the completion of the five year 
followup will provide meaningful information 
concerning the relative effectiveness of the 
methods of therapy under study. 

Diagnostic culdoscopy, gynecography, explo- 
ratory laparotomy, culdoscopic photography 
and culdoscopic ovarian biopsy have all been 
used to compare the usefulness of culdoscopy 
and gynecography, to develop a technique of 
more exact and dynamic culdoscopy and to 
compare the usefulness of the various culdo- 
scopic instruments available. Culdoscopy and 
gynecography have been compared in 44 pa- 
tients and the accuracy and capabilities of each 
technic noted. An intraperitoneal calibrated 
probe has been developed which permits manip- 
ulation, palpitation and accurate mensuration 
of the pelvic organs. Techniques of photography 
and ovarian biopsy have been utilized to give 
permanent gross and microscopic specimens of 
the ovaries. 

Pergonal (human menopausal urinary gona- 
dotropic) is being utilized as an ovarian stimu- 
latory agent in a development of an ovarian 
stimulation test. 24-hour urinary estrogens, 
testosterone production rates and delta 4 an- 
drostine-dion secretory rates are being meas- 
ured. This test will prove useful in the diag- 
nosis of amenorrhea and in the study of ovar- 
ian function. However, it will also be of value 
in the diagnosis and study of endocrinologi- 
cally active ovarian neoplasms. 

Induction of ovulation in anovulatory women 
with Pergonal and human chorionic gonadotro- 



pins has been possible but has been relatively 
unsatisfactory because of the 40% of multiple 
pregnancy rate. Utilizing patients studied as a 
part of the ovarian stimulation test studies, 
dosage regimens of these two agents are being 
evaluated. The objective of the study is the se- 
lection of dosage regimes most effective for 
induction of consistent, single ovulations in 
anovulatory women. 

Urine oxygen tension (pOa) has been studied 
in dogs under standard conditions of anesthe- 
sia, temperature, and urine sampling in both 
hydropenia and water diuresis. Osmotic diu- 
resis in hydropenia and free water clearance 
uniformly drops pOa. This suggests that pOz is 
related to sodium reabsorption in the loop of 
Henle, i.e., that increased sodium reabsorption 
leads to increased oxygen consumption in the 
renal medulla, and therefore, to a fall in urine 
pOj. Studies with ethacrynic acid which blocks 
the sodium pump in the loop of Henle would ap- 
pear to support this theory. 

In various experiments, rats and guinea pigs 
have been transplanted with excessive numbers 
of histocompatible endocrine tissues in order 
(1) to study the ability of normal tissue to re- 
duce its synthetic activity and (2) to magnify 
small physiological effects of normal amounts 
of transplanted tissue and thus clearly define 
their activity. By using excessive numbers of 
transplanted isologous parathyroid glands, we 
have found that a state of obligatory hyper- 
parathyroidism results. On the other hand, by 
using up to 30 transplanted pituitaries in a sin- 
gle hypophysectomized recipient, we have been 
able to demonstrate trophic activity from 
transplanted tissue proportional to the dose 
transplanted and much higher than previously 
observed. Similarly, 10 to 20 transplanted ani- 
mals have been demonstrated to reverse the 
effect of pinealectomy in female rats. 

A combination antimetabolite-metabolite 
program of therapy is being developed to ad- 
minister systemic chemotherapy for dissemi- 
nated neoplasms while protecting the anti 
metabolite. Previous combinations of such drugs 
have been given and usually in the ratio of 1 to 
1 or 2 to 1 with the metabolite being given in 
very high concentrations intramuscularly or 
intravenously. Since very little lasting benefit 



NATIONAL HEART INSTITUTE 



67 



has been obtained in human neoplasms from 
such a combination of drugs, it is proposed 
that the metabolite when given in this high 
concentration voids or blocks the effect of the 
antimetabolite on the cancer cell as well as in 
susceptible tissues such as the marrow and gas- 
trointestinal tract. Because of the slow dou- 
bling time of most human neoplasms, prolonged 
exposure of a cancer cell population to the anti- 
metabolites is a logical approach to chemo- 
therapy. Such an exposure for several weeks 
may be feasible, utilizing intra-arterial protec- 
tive specific metabolites on normal rapidly pro- 
liferating cell populations susceptible to toxic- 
ity. Encouraging toxicity studies in primates 
have been underway during the past year, pro- 
tecting the iliac marrow by infusion of the 
metabolite into the superior gluteal artery. Pi- 
lot studies are now proposed which will deter- 
mine the clinical feasibility of this project. 

Ethiodol is a commonly used lymphangio- 
graphic radio-opaque medium. Preliminary 
carcinogenesis studies in mice suggested that 
this agent might have activity which would dis- 
courage its further use. These animal studies 
utilized Ethiodol. Subsequent animal investiga- 
tions, comparing the poppy seed oil component 
of Ethiodol, with Ethiodol itself and other sim- 
ilar agents are now being followed in several 
strains of mice as well as in rats and dogs. Pre- 
liminary data suggests that the poppy seed oil, 
as previously reported, is carcinogenic in cer- 
tain mouse strains. 

A study of tumor growth and metastases 
with transplanted, induced and spontaneous tu- 
mors in mice has shown that metastases ob- 
served in these three separate tumor systems, 
in both amputated and intact tumor-bearing 
mice was similar even though the growth rates 
of the tumor systems varied markedly. The sim- 
ilarity in the extent of metastases is believed 
to be due to prolonged survival which was per- 
mitted by individual animal housing. On the 
basis of this study, it appears that three tumor 
systems evaluated might be interchangeably 
used in metastases studies if recognition is 
to their varied growth rates. 

Induced and spontaneous tumor systems 
were studied to determine if the rate of tumor 
growth and the number of metastases would be 
altered if a second tumor was transplanted into 



the host after the initial tumor had been re- 
moved. We observed that the tumors implanted 
after the initial tumor had been removed, grew 
in exactly the same manner as they did in con- 
trol animals even when metastases from the 
metastatic tumor were extensive. 

In a controlled study using inbred mice 
and transplantable tumor systems, incisional 
biopsy of the tumor was shown to increase sig- 
nificantly the incidence of pulmonary metas- 
tases. It was observed in one tumor system that 
more animals possessed small foci of tumor in 
their lungs four weeks after their primary tu- 
mor was removed than would be expected to 
die of pulmonary metastases. These foci were 
infiltrated with mononuclear cells and cells in 
mitosis, suggesting that the tumor cells were 
being rejected by the host. 

Studies have been carried out to determine 
the role of the lymphoreticular tissues in chem- 
ically-induced tumor immunity. Splenectomy 
prior to tumor inoculation had no effect on tu- 
mor immunity. Splenectomy performed three 
weeks after tumor inoculation, however, re- 
moved most of the animal's resistance to 
further tumor challenge. Regional lymphaden- 
ectomy had the opposite effects. When lympha- 
denectomy was performed prior to tumor 
inoculation, host resistance was markedly 
depressed but when performed after the tumor 
was growing, no effect on tumor growth was 
found. Adoptive transfer of chemically-induced 
tumor immunity was possible when large doses 
of immune spleen or lymph node cells were 
given intraperitoneally one week prior to tumor 
inoculation. Further studies are now underway 
to investigate whether the adoptively trans- 
ferred immune cells are competant within dif- 
fusion chambers, and whether immune RNA is 
responsible for the transference of tumor im- 
munity. 

Studies have been performed to investigate 
the nature of the immunity imparted by cer- 
tain chemically-induced tumors upon their 
hosts. Particular emphasis has been focused on 
whether chemically-induced tumor immunity 
exists in the autochthonous host, and whether 
the specific immunity that we see in later 
transplant generations is due to antigenic al- 
teration of the tumor in serial passages. The 



68 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



cellular localization of this antigen seems to be 
in the nuclear fraction of the tumor tissue. 
With weakly antigenic tumors, i.e., S-91 mel- 
anoma, prior immunization with cell-free tu- 
mor extracts had no significant effect on tumor 
growth. Enhancement occurred when this ex- 
tract was given at the time of tumor inocula- 
tion. 

The Surgery Branch has been pleased to co- 
operate with the Precious Blood High School of 
Holyoke, Massachusetts in having five of their 
students spend time with us in preparation for 
their student science projects. This work was 
directed toward experimental metastasis in 
mouse tumor systems. Instruction, direction 
and limited equipment and animals were sup- 
plied to these students in order that their stud- 
ies could be completed back at their school. 
Carrying out separate projects, both in 1963 
and 1964, all five have won awards and cash 



prizes from their school, the American Cancer 
Society, and state-wide awards sponsored by 
Massachusetts Institute of Technology. 

Four Clinical Associates received recognition 
and prizes for their work. Drs. Riggins and 
Pilch received first prize for their paper on bas- 
ic studies on the antigenicity of fibrosarcoma, 
from the American Academy of Orthopaedic 
Surgeons. Dr. John Minton was given the 
James Ewing Society's Resident Award for his 
work on laser radiation on animal tumor sys- 
tems, and Dr. Ruben Gittes won a first prize 
from the American Urological Association for 
his paper on transplanted isologous parathy- 
roid glands as an experimental model for the 
study of hyperparathyroidism. All of these 
papers were judged best in their respective 
groups in competition with surgical residents 
from medical centers all over the United 
States. 



NATIONAL HEART INSTITUTE 



LABORATORY OF BIOCHEMISTRY 

Section on Enzymes 

Eesearch in this laboratory continues to in- 
volve studies on a number of fundamental 
problems that provide opportunities to eluci- 
date biochemical mechanisms hitherto unex- 
plored. Major areas of investigation include 
the following: (1) the regulation of divergent 
biosynthetic pathways of metabolism; (2) the 
metabolism of one carbon compounds; (3) the 
metabolism of amino acids; (4) cystathionine 
synthesis and transsulf uration ; (5) the mech- 
anism of action of vitamin B12 coenzyme; (6) 
the anaerobic hydroxylation of aromatic com- 
pounds ; (7) the uptake of amino acids by cellu- 
lar membrane preparations and (8) the dissim- 
ilation of heterocylic compounds. Several of 
these studies have inevitably embraced broad 
areas of enzyme function in metabolism such 
as those concerned with the roles of vitamin B12 
derivatives and of non-heme iron electron car- 
rier proteins (i.e., ferredoxin) . 

The Regulation of Divergent Biosynthetic 
Pathways of Metabolism 

Cumulative Feed-back Inhibition op 
Glutamine Synthetase. Previous studies in 
this laboratory showed that the glutamine 
synthetase of Escherichia coli is uniquely inhib- 
ited by eight different metabolites whose 
synthesis may involve glutamine as a precur- 
sor. Whereas the inhibition of glutaminesynthe- 
tase activity by any single metabolite is only 
10-40 percent, when several or all inhibitors 
are present simultaneously, an over-all cumula- 
tive inhibition is observed that would be ex- 
pected if each inhibitor is independent in its 
action and is uninfluenced by the presence of 
the others. When all eight end metabolites are 
present simultaneously, over 95 percent inhibi- 
tion of the enzyme is observed. 



In order to determine the mechanism of this 
unusual type of feed-back regulation, the gluta- 
mine synthetase is being studied in detail. 
After a 200-fold purification from extracts of 
E. coli, the enz3Tne was isolated as a pure ho- 
mogeneous protein. It has a molecular weight 
of approximately 900,000, as estimated by its 
sedimentation rate in a sucrose density gra- 
dient, and it migrates as a single major band 
during gel electrophoresis. Treatment of the 
enzyme with 1 M urea and 0.01 M EDTA re- 
sults in its quantitative disaggregation to enzy- 
matically inactive subunits of approximately 
100,000 molecular weight. In the presence of 
manganous salts the subunits reaggregate to 
form the native molecule and the enzyme acti- 
vity is restored. 

An explanation for the fact that only partial 
inhibition is observed at saturating concentra- 
tions of each inhibitor was sought through a 
more detailed kinetic analysis of the reaction 
catalyzed by the purified enzyme. The data 
obtained with some of the inhibitors are con- 
sistent with the hypothesis that the enzyme 
contains at least two binding sites for the inhib- 
itors. Binding at one site occurs at relatively 
low inhibitor concentration and results in inhi- 
bition of the enzyme. With increasing inhibitor 
concentration binding occurs also at a second 
site (activator site) and results in partial reac- 
tivation of the inhibited enzyme, possibly by 
reducing the affinity of the enzyme for the inhib- 
itor at the inhibitor site. 

In view of the central role of glutamine 
synthetase in nitrogen metabolism and because 
of its remarkable susceptibility to cumulative 
feed-back inhibition by a large variety of end 
metabolites, it provides an excellent model 
system to study manifold regulation of diver- 
gent metabolic pathways. In addition, its high 
degree of stability and unusual physical prop- 
erties offer opportunities to investigate sever- 
al interesting aspects of protein chemistry ; i.e.. 



69 



70 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the mechanism of its aggregation and disag- 
gregation. Future experiments will be directed 
at a more intensive investigation of the pu- 
riiied protein in relation to its fine structure 
and feed-back characteristics. 

One Carbon Metabolism 

Methane Fermentation. Previous stud- 
ies in this laboratory have shown that cell- 
free extracts of Methanocarcina barkeri cata- 
lyze the reduction of methanol, CO2 and 
methyl-Bi2 to methane. For this reduction, 
molecular hydrogen serves as the electron do- 
nor and ATP and CoA are required. Further 
studies have shown that cell-free extracts cata- 
lyze the accumulation of methyl-Bi2 when incu- 
bated with substrate levels of reduced vitamin 
B12 (i.e., Bi2s) methanol, ATP and CoA. 

Labeled methyl-Bi2 has also been isolated 
after incubation of crude extracts with carbon- 
labeled methanol and unlabeled methyl-Bjo 
under conditions where both substrates are re- 
duced to methane. However in the latter exper- 
iment the amount of labeled carbon trapped in 
the residual pool of methyl-Bi2 was consider- 
ably less than would be expected if free meth- 
yl-Bi2 is an obligatory intermediate in methane 
formation. It is concluded that enzyme-bound 
methyl-Bi2 or a related derivative, but not free 
methyl-Bi2 is probably an intermediate in 
methane formation. 

Synthesis of Acetate from CO,. Investi- 
gations on the enzymic condensation and re- 
duction of CO2 to acetate have been continued. 
The possibility that methyl-Bi2 is an interme- 
diate in this novel biochemical process was sug- 
gested by earlier experiments showing that 
cell-free extracts of Clostridium therrtioaceti- 
cum catalyze the conversion "C-methyl-Bi2 to 
methyl labeled acetate in the presence of car- 
bon dioxide, CoA and pyruvate. A role of 
methyl-Bi2 in acetate synthesis is further sup- 
ported by recent experiments showing that ^^C- 
methyl-Bi2 can be isolated from incubation mix- 
tures that contained initially "CO2, unlabelled 
methyl-Bi2, pyruvate, CoA and crude enzyme 
preparation. Additional supporting evidence' 
on this point is provided by recent exper- 
iments showing that cell-free extracts of C. 
thermoaceticuin catalyze the de novo synthesis 



of methyl-Bi2 from CO2 and reduced vitamin 
B12. 

In order to determine the mechanism by 
which methyl-Bi2 is converted to acetate, 
efforts have been made to purify the enzyme 
system catalyzing this conversion. By chroma- 
tography on DEAE cellulose, cell-free extracts 
were resolved into three protein fractions, all 
of which are required in combination in order 
to catalyze the conversion of methyl-Bi2 to ace- 
tate. One of the three protein fractions has 
been identified as ferredoxin. It was inciden- 
tally discovered that extracts of Clostridium 
sticklandii that are unable to catalyze the for- 
mation of acetate from CO2 and methyl-Bia, are 
able to do so if they are supplemented with one 
of the unidentified protein fractions from C. 
thermoaceticum. 

These results indicate that the reduction of 
CO2 to acetate is a unique biochemical process 
that involves pathways of one carbon metabo- 
lism hitherto unrecognized. 

The Metabolism of Amino Acids 

Reductive Deamination. The reductive 
deamination of glycine and the fermentation of 
lysine to form fatty acid derivatives were pre- 
viously shown to be coupled with the esterifica- 
tion of orthophosphate to form ATP. Various 
substances, including DPNH, dimercaptans 
and reduced methyl viologen, are able to serve 
as electron donors in these reactions, and avail- 
able evidence indicates that the phosphoryla- 
tion may be associated with the electron trans- 
port process rather than substrate alterations. 
These reductive deaminations therefore appear 
to offer unusual opportunities to investigate 
the mechanisms of anaerobic oxidative phos- 
phorylation in completely soluble enzyme sys- 
tems. Accordingly, efforts have been made to 
isolate and purify the enzymes from Clostrid- 
ium sticklandii that are involved in reductive 
deamination per se and also those involved in 
the anaerobic electron transport chain of this 
organism. 

An acidic, low molecular weight protein com- 
ponent of the glycine reductase system, desig- 
nated as protein A, has been purified 500 to 700 
fold and some of its properties have been stud- 
ied. Its molecular weight is approximately 



NATIONAL HEART INSTITUTE 



71 



20,000. It is moderately heat stable, tends to 
aggregate with concomitant loss of catalytic 
activity, and is sensitive to inactivation by ex- 
posure to ultraviolet light (360-400 m^t). The 
latter fact suggests the presence of a light-sen- 
sitive chromophore absorbing in the yellow 
region of the spectrum. Protein A is appar- 
ently not involved in the terminal phosphory- 
lation reaction since it catalyzes neither the ex- 
change of orthophosphate with ATP nor of 
ADP with ATP. It is distinct and is readily 
separable from the menadione-dependent p-ni- 
trophenyl phosphatase previously discovered in 
extracts of C. stricklandii. 

In addition to protein A, five other proteins, 
appearing to have electron carrier functions 
have been highly purified from extracts of C. 
sticklandii and partially characterized. One of 
these is the non-heme iron electron carrier pro- 
tein, ferredoxin, that is an essential component 
of the system catalyzing the reductive deami- 
nation of glycine and also of the multi-enzyme 
system catalyzing the reductive cleavage of pro- 
line to form A-aminovaleric acid. The ferre- 
doxin from C. sticklandii is similar in size and 
acidity to ferredoxins isolated from other or- 
ganisms and will satisfy the ferredoxin re- 
quirements for other enzymatic reactions; i.e., 
the sjTithesis of acetate by C. tkermoaceticum 
and the reduction of DPN by molecular hydro- 
gen by extracts of C. kluyveri. 

Of the four other electron-carrier proteins 
isolated, two are flavoproteins that catalyze the 
oxidation of reduced pyridine nucleotides. Both 
are acidic proteins with molecular weights of 
about 25,000. One of these flavoproteins is a 
TPNH specific dehydrogenase; the other is a 
DPNH specific dehydrogenase. They catalyze 
the transfer of electrons from the respective 
reduced pyridine nucleotides to oxygen, or any 
of a number of artificial electron acceptor dyes. 
Their role in the electron transport system of 
C. sticklandii is indicated by the fact that, in 
the presence of their respective reduced pyri- 
dine nucleotides, they catalyze the reduction of 
two other electron acceptor proteins that have 
been partially purified from the cell-free ex- 
tracts. One of these is characterized by its 
orange-red color which is lost when it is re- 
duced enzymatically or treated with sodium bo- 
rohydride or hydrosulfite. It is a small (MW, 



6000-7000) acidic protein with a light absorp- 
tion band having a maximum at 490 m/x. The 
other electron acceptor protein is red-violet in 
color, becoming bleached upon enzymic or 
chemical reduction. It too is a small (MW, ca. 
25,000) protein having an absorption band at 
535 m/i. The reduced forms of both of the elec- 
tron carrier proteins are readily autoxidizable 
and revert to their characteristic oxidized color 
when exposed to oxygen. Although the precise 
roles of these proteins are not known, in view 
of their electron carrier capacities it is reason- 
able to assume that they are a part of the an- 
aerobic electron transport chain present in this 
organism. Further studies are aimed at the 
characterization of their prosthetic groups and 
the establishment of their biological function. 

Incidental to the purification of the above 
proteins, was the purification also of the qui- 
none-dependent p-nitrophenyl phosphatase. This 
curious enzyme was previously discovered in 
extracts of C. sticklandii but resisted efforts to 
purify it by the conventional fractionation 
procedures then available. It has now been ob- 
tained in a more highly purified form as a by- 
product of the above studies. The mechanism 
of its action will be re-explored using P^^-la- 
beled p-nitrophenyl phosphate which is now 
commercially available. 

Cystathionine Biosynthesis and Trans-Sulfura- 
tion 

Cystathionine y-SYNTHETASE. Cystathi- 
onine 7-synthetase catalyzes a reaction be- 
tween L-cystein and o-succinyl-L-homoserine to 
form L-cystathionine and succinate. This en- 
zyme has been isolated from Salmonella ex- 
tracts as a pure homogeneous protein. The pu- 
rified enzyme has a molecular weight of 
160,000±5,000 and contains 4 moles of pyri- 
doxal phosphate per mole. When treated with 
guanidine and jS-mercaptoethanol at pH 7.0 it 
is resolved into 4 identical subunits 
(MW=43,000). Kinetics of the enzymatic reac- 
tion have been studied in detail. The Km's for 
o-succinyl-L-homoserine and L-cysteine are 7 
X 10-= M and 7 X IQ-^M, respectively. The 
product of the reaction, L-cystathionine, was iso- 
lated as a pure crystalline solid and was posi- 
tively identified by direct comparison with an 



72 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



authentic sample of L-cystathionine. The enzy- 
matic product and the authentic sample were 
identical with respect to chromatographic be- 
havior, appearance in light, phase contrast, 
dark field and polarized light microscopy, 
infra-red spectra, optical rotary dispersion, 
chemical reactivity and enzymic activity. 

The purified cystathionine-y-synthetase also 
catalyzes a y-elimination reaction in which o- 
succinyl-L-homoserine reacts with water to 
form succinate, NH3 and a-keto-butyrate. The 
Km for o-succinyl-L-homoserine in this reac- 
tion is 2.8 X 10-* M. 

Gamma Elimination Reactions. The 
conversion of cystathionine to cysteine, NH3 
and a-ketobutyrate is a prototype for several 
enzyme-catalyzed y-elimination reactions. In- 
sight into the mechanism of this reaction was 
afforded by the discovery that N-ethylmalei- 
mide is able to react with a transient interme- 
diate produced in the cleavage of cystathionine 
to form a derivative that contains the elements 
of maleimide and the 4-carbon chain of the 
substrate; other products of the reaction are 
cysteine and NH3. The structure of the malei- 
mide derivative was shown to be: 

CHj 
H-C-COCOOH 



-0° 



K 

The reaction with maleimide represents a 
new type of pyridoxal — P catalyzed reaction, 
that can be described as a y-elimination 
coupled to Michael addition of a j8-carbanion to 
an electrophilic double bond. 

The use of maleimide appears to provide a 
new chemical approach to the enzyme-bound 
tautomeric intermediate thought to participate 
in pyridoxal phosphate catalyzed reactions. 

Consequent to the widespread use of N-eth- 
ylmaleimide as a protein sulfhydryl trapping 
agent, it has become clear that this reagent can 
also react with amino groups. The nature of 
the latter reaction has not previously been es- 
tablished. In collaboration with Dr. Norman 
Sharpless (NIAMD) the products of reaction 
with a variety of amines and amino acids have 
been isolated, and their structures determined. 
In every case the reactions have been found to 



involve amino group addition to the double 
bond of maleimide. The reaction of proline is 
interesting because of its rapidity at physiolog- 
ical pH, and because the product reverts to 
starting materials at pH 5, and more slowly in 
strong acid. 

Distribution of Trans-sulfuration 
Enzymes The distribution of enzymes cata- 
lyzing the various kinds of trans-sulfuration 
reactions has been examined in several differ- 
ent organisms in an effort to discover possible 
evolutionary relationships. The transfer of sul- 
fur from homocysteine to make cysteine is the 
consequence of two coupled reactions: (1) a 
reaction of serine and homocysteine to form 
cystathionine and (2) a reaction in which cys- 
tathionine is cleaved to form cysteine, a-keto- 
butyrate and ammonia. Enzymes catalyzing 
these two reactions are present in fungi and in 
mammalian liver; they have not as yet been 
detected in bacteria. In contrast, bacteria con- 
tain two different enzymes; (1) an enzyme cat- 
alyzing the synthesis of cystathionine from 
succinyl-o-homoserine and cysteine, and (2) an 
enzyme that catalyzes cleavage of cystathio- 
nine to form homocysteine, NH3 and pyruvate. 
Together, these two enzymes catalyze the 
transfer of sulfur from cysteine to homocys- 
teine. Only one of these latter enzymes, the 
cystathionine cleavage enzyme, could be found 
in fungi; neither enzyme has been detected in 
mammalian tissues. 

Mechanism of Action of Vitamin B12 Coenzyme 

Ethanolamine Deaminase. An enzyme 
catalyzing the vitamin B12 dependent deamina- 
tion of ethanolamine to form acetaldehyde is 
the subject of a continuing investigation. The 
enzyme has been purified 80-fold from extracts 
of an ethanolamine fermenting Clostridium, 
previously isolated in this laboratory. A highly 
sensitive assay procedure for this enzyme was 
developed in which the acetaldehyde formed 
from the enzymatic deamination of ethanola- 
mine is reduced to ethanol in the presence of 
added alcohol dehydrogenase and DPNH. The 
oxidation of DPNH associated with the acetal- 
dehyde reduction is measured spectrophotomet- 
rically. The partially purified enzyme is rela- 
tively unstable ; it loses half of its activity over a 



r 



NATIONAL HEART INSTITUTE 



73 



24 hour period at 0°. Partial protection against 
inactivity is achieved by adding ethanolamine. 
Preliminary studies with sucrose gradient sedi- 
mentation indicate the enzyme has a molecular 
weight of about 250,000. When obtained in a 
relatively pure form, this enzyme will be used 
for more detailed studies on the mechanism of 
action of vitamin B12 coenzymes. 

Anaerobic Hydroxylation of Aromatic Com- 
pounds 

Hydroxylation of Phenylalnine and 
Nicotinic Acid. The discovery that Clostrid- 
ium glycolicum requires phenylalanine but 
not tyrosine for growth, and the demonstration 
that 6-hydroxynicotinic acid is an early in- 
termediate in the anaerobic dissimilation of nic- 
otinic acid by a Clostridium, sp., suggested 
that these organisms might have the capacity 
to catalyze anaerobic hydroxylation of aromat- 
ic compounds. The hydroxylation of aromatic 
compounds has hitherto been regarded as an 
obligatory oxygen-dependent process and 
therefore restricted to aerobic organisms. 

Encouraged by previous results showing that 
isotopically labeled tyrosine is present in the 
protein of C. glycolicum, when grown on a me- 
dium containing uniformly labeled phenylanine, 
efforts were made to demonstrate the con- 
version of phenylanine to tyrosine by cell sus- 
pensions. Although cell suspensions catalyze 
the anaerobic decomposition of phenylalanine 
to a variety of substances, tyrosine could not be 
identified among the products. In order to ob- 
viate ambiguities inherent in the previous 
results obtained with uniformly labeled phenyl- 
alanine, studies are now in progress to deter- 
mine if isotope from ring-labeled phenylalanine 
is incorporated into protein during the growth 
of C. glycolicum. 

The enzyme from the Clostridium sp. that 
catalyzes the hydroxylation of nicotinic acid 
was partially purified and some of its proper- 
ties were determined. It catalyzes a reaction 
between TPN and nicotinic acid to form TPNH 
and 6-hydroxynicotinic acid. The reaction can 
be followed spectrophotometrically by measur- 
ing the absorbancy at 340 m/i due to TPNH ac- 
cumulation. The reaction requires the presence 
of ferrous sulfate and glutathionine and is 



markedly stimulated by the presence also of 
orthophosphate or inorganic arsenate. Other 
salts cause little or no stimulation. The stabil- 
ity of the enzyme at 0° and —IOC is uniquely in- 
fluenced by various salts. Thus at —10° stability 
in the presence of 0.1 M salts decreases in the 
following order: KHP04(pH7.3) >Tris. Cl> 
NaCl> LiCl> KC1> NH4C1> imidazole CI. 
However, at 0° C the order is very different; 
i.e., KHP04> NH4C1> KC1> NaCl> Tris Cl> 
LiCl> imidazole CI. Further purification of the 
enzyme is in progress to determine if it contains 
an unusual prosthetic group that could help to 
explain its unique biochemical function. 

The Uptake of Amino Acid by Cellular Mem- 
brane Preparations 

Isolated cytoplasmic membranes from wild 
type E. coli, strain W and from a mutant 
strain WS that is deficient in the ability to 
transport glycine, are being studied in order to 
determine the mechanism of carrier-mediated 
transport of amino acids. The membrane 
preparations are prepared by osmotic shock of 
protoplasts. 

Chemical and enzymatic determinations 
combined with electron microscopy show that 
these membrane preparations have little or 
none of the cytoplasmic constituents of intact 
cells. Thus, they contain less than 1% of the 
total cellular DNA and RNA and only 20% of 
the total cellular protein. Furthermore the iso- 
lated membranes have morphological charac- 
teristics, as seen in the electron microscope, of 
a bimolecular lipid leaflet with a thin layer of 
external material. 

Glycine uptake by membranes of the wild 
type strain is a hyperbolic function of the ex- 
ternal glycine concentration, approaching satu- 
ration at external concentrations of 5 fig per 
ml ; this suggests that a carrier mediated proc- 
ess is involved. This conclusion is further sup- 
ported by the fact that glycine uptake is depend- 
ent upon an energy source (i.e., glucose or 
ATP or DPNH or TPNH or succinate, etc.) ; it 
is inhibited by a variety of metabolic inhibitors 
some of which are known to inhibit oxidative 
phosphorylation ; it is inhibited by alanine, ser- 
ine and threonine but not by other amino acids ; 



74 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



alanine, serine and threonine but not other 
amino acids, added to the suspended medium 
cause rapid exchange of glycine previously tak- 
en up by the membranes; uptake is markedly 
influenced by temperature. 

In contrast, the uptake of glycine by mem- 
brane preparations of the mutant strain WS is 
a linear function of the external concentration 
over a broad range (0-10 /ig per ml), and is 
relatively insensitive to temperature (0-37° 
C) ; it is non-energy dependent and is not in- 
fluenced by metabolic inhibitors or other amino 
acids. 

These results indicate that membrane prepa- 
rations are promising material for studies on 
the mechanism of carrier-mediated transport 
of amino acids. In future studies, efliorts will be 
made to (1) establish the chemical nature of 
the intracellular exchangeable and nonex- 
changeable amino acid pools, (2) to determine 
the nature of the energy-linked process in- 
volved in transport and (3) to elucidate the 
character of the functional defect in the mu- 
tant strain WS. 

The Metabolism of Heterocyclic Compounds 

Chemistry of Riboflavin. To facilitate 
enzyme studies on the metaboli of heterocyclic 
compounds and also to explore techniques that 
may be applicable to projected studies on free 
radical intermediates in electron transport proc- 
esses, investigations are being made on the 
chemistry of riboflavin and related isoalloxa- 
zine derivatives. Hydroxyethylflavin was ob- 
tained by periodate degradation of the ribityl 
group followed by borohydride reduction. 
When hydroxyethylflavin was treated with 
thionyl chloride, a facile cyclization involving 
the hydroxyethyl side chain and the N-atom at 
position 1 of the isoalloxazine ring occurred. The 
structure of the cyclized product was deduced 
from NMR, ultraviolet and visible spectral data. 
The compound was quite unstable in aqueous 
base, probably undergoing cleavage of the py- 
rimidine ring. Like other isoalloxazine com- 
pounds, the cyclized product could be reduced by 
zinc and acid to give a semiquinone as observed 
by ESR spectroscopy. Riboflavin also reacted 
with thionylchloride to give a compex mixture of 
products that were not fully characterized. How- 



ever, 6,7- dimethyl - 8 - hydroxy - ethyllumazine 
a hetercyclic ring system resembling that of 
hydroxyethyl flavin but which lacks the benze- 
noid ring characteristics, failed to undergo 
similar cyclization even under more drastic con- 
ditions. 

Electron spin resonance (ESR) spectra of 
semiquinones of lumiflavin, hydroxyethylflavin 
and cyclized hydroxyethylflavin all showed 
characteristic hyperfine splittings. A tentative 
interpretation of the lumiflavin semiquinone 
spectrum was developed. 

Nicotinic Acid Degradation. One of the 
steps in the anaerobic degradation of nicotinic 
acid by a Clostridium sp. is the conversion of 
6-oxo-l,4,5,6-tetrahydronicotinic acid to a-meth- 
ylene glutaric acid. To study this enzymic 
step in detail efforts were made to develop a 
practical chemical synthesis of the 6-oxo com- 
pound for use as a substrate. Three independ- 
ent routes of synthesis were attempted which 
involved (1) the intermediary synthesis of an 
a-pyrone derivative through condensation of 
ethyl formyl acetate with ethyl aery late; (2) 
The intermediary synthesis of a-cyanoglutarate 
by condensation of ethylcyanoacetate and ethyl 
aery late; and (3) the intermediary synthesis 
of an imino ether by reaction of diethyl-a- 
cyanoglutarate with ethanol and hydrogen 
chloride. Only the latter method yielded the de- 
sired intermediate which was characterized by 
mass spectrum and its IR and NMR spectra. 
Treatment of the imino ether with potassium 
borohydride gave a neutral mixture containing 
two major components. One was identified as 
a-ethoxycarbonyl glutarate. The other has not 
been characterized. 

Section on Comparative Biochemistry 

The activities of the Section on Comparative 
Biochemistry are concentrated on the investiga- 
tion of several basic biochemical problems re- 
lated to lipid metabolism. The discovery in this 
laboratory of the acyl carrier protein (ACP), 
which appears to play a central role in lipid 
metabolism, has laid the foundation of many 
new experiments probing the interactions of 
lipids and proteins. Study of the mechanism of 
involvement of ACP in fatty acid biosynthesis 
and complex lipid biosynthesis has allowed the 



NATIONAL HEART INSTITUTE 



75 



elucidation of the enzymatic mechanism of these 
reactions. Knowledge of the individual reactions 
has led to new studies on regulatory mecha- 
nisms. Study of ACP itself has led to the eluci- 
dation of its active site, a prosthetic group, 
4'-phosphopantetheine. The finding of 4'-phos- 
phopantetheine as a component of a protein 
has led to a search for other proteins with sim- 
ilar prosthetic groups. In addition, interest has 
now focused on the general significance and 
mode of biosynthesis of such pantothenate- 
containing proteins. 

Structure and Substrate Binding Site of ACP 

All of the reactions of fatty acid synthesis in 
E. coli occur with the substrates bound in thio- 
ester linkage to the single sulfhydryl group of 
ACP (see below) . The nature of the substrate 
binding site was studied by labeling this site 
eii2;ymatically to form C"-malonyl-S-ACP and 
then subjecting the protein to peptic hydro- 
lysis. Several C"-peptides were isolated by 
Dowex-1 column chromatography and paper 
electrophoresis. Each of these peptides was 
shown to contain unimolar amounts of 
2-C^*-malonate, 2-mercaptoethylamine, ,8-ala- 
nine, pantoic acid, and phosphate, as well as a 
variety of amino acids. Demonstration of the 
pantoic acid required treatment of the peptides 
with Pronase (a proteolytic enzyme) followed 
by gas-liquid chromatography, whereas dem- 
onstration of the other components was ac- 
complished by routine analyses. Although 
2-mercaptoethylamine, /3-alanine, pantoic acid 
and phosphate are important components of 
CoA, ACP peptides lacked pyrophosphate, ri- 
bose and adenine, indicating that the substrate 
binding site of ACP resembled, but was not the 
same as, CoA. Hydrolysis of ACP in 3N NaOH 
at 102° yielded 4'-phosphopantothenic acid, 
which, after treatment with E. coli alkaline 
phosphatase, yielded equivalent amounts of 
inorganic phosphate and pantothenic acid (as- 
sayed microbiologically with Lactobacillus ara- 
binosus) . In order to identify the entire pros- 
thetic group intact, ACP was hydrolyzed at pH 
12 (70°) for one hour, thereby liberating 4'- 
phosphopantetheine from the protein. The 4'- 
phosphopantetheine was identified by conver- 



sion enzymatically to CoA with ATP, de- 
phospho CoA pyrophosphorylase and de- 
phospho-CoA kinase. CoA was assayed with 
phosphotransacetylase. These experiments es- 
tablished that 4'-phosphopantetheine is the 
prosthetic group and substrate binding site of 
ACP. 

Further studies utilizing alkaline and enzy- 
matic hydrolysis of this prosthetic group indi- 
cate that 4'-phosphopantetheine is bound in 
phosphodiester linkage to a serine residue to 
ACP. The following compositions have been de- 
termined for two C^*-peptic peptides isolated 
from 2-C"-malonyl-S-ACP: 

(1) Asp, Leu, Ser, 4'-phosphopantetheine 

(2) Ala, Gly, Asp, Leu, Ser, 4'-phospho- 
pantetheine. 

Using the Edman degradation with subtractive 
analysis and also fluorodinitrobenzene the se- 
quences of the two peptides were shown to be 
Asp-Ser-Leu and Gly-Ala-Asp-Ser-Leu, respec- 
tively. Treatment of peptide 2 with mild alkali 
caused j8-elimination of 4'-phosphopantetheine 
from the serine which was converted to dehy- 
droalanine. Subsequent acid hydrolysis led to 
the formation of pyruvic acid (assayed with 
lactic dehydrogenase), thus establishing the 
linkage of 4'-phosphopantetheine to ACP as a 
phosphodiester linkage to the serine hydroxyl 
group. Additional studies of ACP have shown 
that the N-terminal residue is serine and the 
C-terminal residue is alanine. 

The binding site of ACP, 4'-phosphopantethe- 
ine, is the same as that in CoA. The marked 
specificity of the enzymes of fatty acid synthe- 
sis for thioesters of ACP indicates that it is 
not the binding site of ACP but the remainder 
of the structure which accounts for this specific- 
ity and increased activity of ACP thioesters. 
Further studies of the primary and tertiary 
structure of ACP will be needed to delineate its 
mode of action. 

ACP Involvement in Lipid Metabolism 

ACP was initially demonstrated in extracts 
of Clostridium kluyveri and E. coli as a heat- 
stable protein required in the condensation-de- 
carboxylation reaction of fatty acid synthesis. 
Subsequent studies have amplified its central 



76 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



role in fatty acid biosynthesis as demonstrated 
by the following reactions: 

Acetyl-S-CoA + ACP-SH<=iacetyl-S-ACP + 
CoA-SH (1) 

Malonyl-S-CoA + ACP-SH?^malonyl-S-ACP 
+ CoA-SH (2) 

Acetyl-S-ACP + malonyl-S-ACP^acetoacetyl- 
S-ACP + CO2 + ACP-SH (3) 

Acetoacetyl-S-ACP + TPNH + H*±5D(-)- 
/3-hydroxybutyryl 1-S-ACP + TPN+ (4) 

D(— )-;8-Hydroxybutyryl 1-S-ACP :^ crotonyl- 
S-ACP + H,0 (5) 

Crotonyl-S-ACP + TPNH + H^ ^ butyryl- 
S-ACP + TPN^ (6) 

Palmityl-S-ACP + CoA-SH?:± palmityl- 

S-CoA + ACP-SH (7) 

Palmityl-S-CoA -|- H2O ^ palmitic acid 

+ CoA-SH (8) 

As noted in the above scheme, ACP acts as the 
active acyl carrier in all the intermediate steps 
that lead to long chain fatty acid synthesis. 
Reactocons 1 and 2 indicate the transfer of the 
acetyl and malonyl moieties from acetyl-CoA 
and malonyl-CoA to ACP to form acetyl- and 
malonyl-S-ACP, respectively. Acetyl- and mal- 
onyl-S-ACP condense in reaction 3 to form 
acetoacetyl-S-ACP, CO2 and ACP-SH. Acetoace- 
tyl-S-ACP undergoes reduction by TPNH to 
form specifically D ( — ) -/?-hydroxybutyryl-S- 
ACP (reaction 4), and the latter is dehydrated 
to form crotonyl-S-ACP (reaction 5). Croton- 
yl-S-ACP is fkrther reduced by TPNH to form 
butyry 1-S-ACP (reaction 6). In the normal 
process of long chain fatty acid synthesis bu- 
tyryl-S-ACP substituting for acetyl-S-ACP in 
reaction 3, condenses with another mole of mal- 
onyl-S-ACP thereby initiating another cycle 
which eventually leads to the formation of pal- 
mityl-S-ACP. Reactions 1, 2, 4 and 6 had been 
discussed previously. Reactions 3, 5, 7 and 8 
have recently been investigated. 

/3-Ketoacyl-ACP Synthetase : /?-Ketoacyl- 
ACP synthetase catalyzes reaction 3, the con- 
densation of acetyl-S-ACP with malonyl-S- 
ACP to form acetoacetyl-S-ACP. A new assay 



was 



was developed for this enzyme based upon the 
following coupled system : 

Acetyl-S-ACP + malonyl-S-ACP <^acetoacetyl- 
S-ACP + CO2 + ACP-SH (9) 

Acetoacetyl-S-ACP + TPNH + H^^fc>D(-)- 
j8-hydroxybutyryl-S-ACP + TPN+ (10) 

Sum: Acetyl-S-ACP + malonyl-S-ACP + 
TPNH + H+^D(-)-j8-hydroxybutyryl-S- 
ACP + CO, + TPN- + ACP-SH. 

Coupling of /3-ketoacyl-ACP synthetase with j8- 
ketoacyl-ACP reductase (reactions 9 and 10) 
allows the assay of the synthetase by measur- 
ing spectrophotometrically the disappearance 
to TPNH in the presence of excess acetyl-S- 
ACP, malonyl-S-ACP, TPNH and the purified 
reductase. ;6-Ketoacyl-ACP synthetase was 
purified 192-fold by means of DEAE-cellulose' 
chromatography, ammonium sulfate precip- 
itation, filtration on Sephadex G-lOO and 
chromatography on culcium hydroxyapatite. 
The resulting enzyme preparation, which still 
contained malonyl transacylase activity (reac- 
tion 2) , was freed of the latter by a second fil- 
tration on Sephadex G-IOO. This preparation 
was also free of acetyl transacylase activity 
(reaction 1). 

The synthetase has an absolute requirement 
for fatty acyl-ACP and malonyl-S-ACP, and it 
is stimulated by 2-mercaptoethanol and EDTA. 
Neither fatty acyl-CoA nor malonyl-CoA can 
replace the ACP derivatives unless the appro- 
priate transacylase and free ACP-SH are 
added. Michaelis constants for acetyl- and mal- 
onyl-S-ACP are both about 2 x 10-=M. 

The synthetase was shown to be a sulfhydryl 
protein on the basis of its requirement for 
2-mercaptoethanol for maximal activity as well 
as inhibition of activity by low levels of sulfhy- 
dryl poisons such as iodoacetamide. No inhibi- 
tion was obtained with arsenite or cadmium 
chloride, indicating that no functional dithiol 
groups are present in this enzyme. The inhibi- 
tion by iodoacetamide could be prevented by 
prior incubation of the enzyme with acetyl- 
ACP. Acetyl-CoA and malonyl-ACP were un- 
able to prevent inhibition. These results indi- 
cate that acetyl-ACP is bound to the protein in 
such a manner as to prevent alkylation by io- 
doacetamide. Experiments are now being con- 



NATIONAL HEART INSTITUTE 



77 



ducted in order to isolate stoichiometric 
amounts of this enzyme so that the mechanism 
of this reaction can be further investigated. 

Enoyl-ACP-Hydrase. Enoyl-ACP hydrase, 
an enzyme which catalyzes the reversible 
hydration of crotonyl-S-ACP to form D(— )- 
/3-hydroxybutyryl-S-ACP (reaction 5), has 
been purified approximately 20-fold from 
E. coli extracts. This enzyme is totally inactive 
with thioesters of pantetheine, CoA, and N- 
acetyl cysteamine as substrates. The enzyme is 
heat stable. By using D(— )- and L ( + ) 
isomers of jS-hydroxybutyryl-S-ACP, it was 
shown that only the D( — ) isomer is active 
with this enzyme. Since it is known that §- 
ketoacyl-ACP reductase forms only the D(— ) 
isomer also, it is clear that the D(— ) stereoi- 
somer is the one which functions in fatty acid 
synthesis. 

Termination of Fatty Acid Synthesis 
Cycle. Palmityl-S-ACP (or another long 
chain fatty acyl-ACP) is formed at the end of 
the sequence of reactions represented by reac- 
tions 1-6. The conversion of palmityl-S-ACP to 
palmitic acid could occur through direct deacyl- 
ation of palmityl-S-ACP, or by an interme- 
diate acyl transfer from ACP to CoA to form 
palmityl-S-CoA (reaction 7) followed by deacyl- 
ation of palmityl-S-CoA (reaction 8). Evi- 
dence favors the latter pathway since enzymes 
which catalyze both reactions 7 and 8 have 
been demonstrated whereas direct deacylation 
of palmityl-S-ACP is not catalyzed by E. coli 
extracts. 

Phosphatidic Acid Synthesis. The dis- 
covery of the role of ACP as acyl carrier in 
fatty acid synthesis has stimulated reinvestiga- 
tion of other reactions in which acyl thioesters 
of CoA are involved in metabolism. Important 
among these are the reactions which lead to the 
formation of phospholipids and triglycerides. 
Experiments were undertaken to determine 
whether the acyl group linked to ACP at the 
end of chain elongation can be transferred di- 
rectly to three carbon precursors of phospholip- 
ids or if the intermediate formation of acyl- 
CoA is necessary. A particulate enzyme system 
from E. coli was found which catalyzes the 
conversion of ^-glycerophosphate to phosphatid- 
ic acid in the presence of palmityl-CoA. Chem- 
ically synthesized palmityl-ACP could not re- 



place the acyl-CoA derivative as acyl group 
donor in this reaction. Therefore it appears 
that the acyl group of ACP must be trans- 
ferred to CoA prior to its utilization in phos- 
phatidic acid synthesis. An interesting prelim- 
inary observation, in connection with these 
experiments, is that the particulate enzyme 
system which catalyzes phosphatidic acid 
synthesis contains pantothenic acid. 

Involvement of ACP in the Regulation of Fatty 
Acid Biosynthesis 

NONIDENTITY OF ACP AND LiFOGENIN.. 

Studies on the regulation of fatty acid bio- 
synthesis have been continued. Fatty acid bio- 
synthesis is known to be severely depressed in 
liver extracts from starved rats. Dr. Herbert 
Anker (University of Chicago) isolated from 
yeast a polypeptide (lipogenin) which can stim- 
ulate fatty acid synthesis in starved rat liver 
preparations. Evidence suggesting the pres- 
ence of lipogenin in boiled rat liver mitochon- 
dria was also reported. Anker more recently 
reported that yeast lipogenin has physical prop- 
erties similar to E. coli ACP and that ACP 
can substitute for lipogenin in the starved rat 
liver assay system. Similar experiments were 
attempted in this laboratory. However in no 
instance was ACP capable of stimulating fatty 
acid synthesis by the starved rat liver homog- 
enate. Since the assay system was not repro- 
ducible enough in this laboratory, a more direct 
approach to the problem was made. The discov- 
ery that E. coli ACP contains 4'-phosphopan- 
totheine as a prosthetic group allowed a direct 
testing of Anker's most purified lipogenin for 
the presence of a similar prosthetic group. 
Treatment of ACP with strong alkali at 102° 
convert the prosthetic group to free 4'-phos- 
phopantotheine acid which can be converted to 
pantothenic acid with E. coli alkaline phospha- 
tase. The latter can be identified in a micro- 
biological assay. Subjecting purified lipogenin 
to this treatment yieled no pantothenic acid. 
Therefore it is concluded that lipogenin is not 
identical with ACP. It can be argued that lipo- 
genin is similar to ACP except that the pros- 
thetic group is bound through a linkage that is 
not hydrolyzed by alkali, or perhaps lipogenin 
might represent apoACP — i.e., ACP minus the 



78 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



prosthetic group, or that lipogenin does not re- 
quire a prosthetic group. None of these possibil- 
ities seem probable. 

Feedback Inhibition. Recent reports 
from a number of laboratories have indicated 
that fatty acid synthesis is sensitive to feed- 
back inhibition by long chain acyl-CoA deriva- 
tives such as palmityl-CoA. One site of feed- 
back inhibition is at the level of malonyl-CoA 
synthesis by acetyl-CoA carboxylase. At least 
one other enzyme of the six enzymes required 
for fatty acid synthesis from acetyl-CoA and 
malonyl-CoA in E. coli is subject to severe 
feedback inhibition. The enzyme, /?-ketoacyl- 
ACP reductase, is competitively inhibited by 
very low concentrations of palmityl-CoA. The 
interaction of this enzyme and compounds 
which act as feedback inhibitors is under 
study. In connection with this work the obser- 
vation has been made that many apparently 
unrelated enzymes are inhibited by palmityl- 
CoA. The physiological significance of inhibi- 
tion by long chain acyl-CoA derivatives in gen- 
eral is being evaluated. 

Biosynthesis of ACP 

The demonstration that ACP contains 
4'phosphopantetheine as a prosthetic group has 
opened the question of the biological synthesis 
of holoACP. It is clear that the prosthetic 
group is linked through phosphodiester linkage 
to a serine hydroxyl of the protein. In order to 
study the mechanism of synthesis, apoACP is 
required. Several approaches have been at- 
tempted to obtain this. Chemical hydrolysis of 
the prosthetic group from the protein has been 
unsuccessful because the procedure destroys 
the serine residue. All available known phos- 
phatases have been tested, but none so far has 
been found to remove the prosthetic group 
from ACP. The most recent approach, and the 
one most likely to succeed, is production of 
apoACP by an E. coli mutant which cannot 
make pantothenic acid. By growing this orga- 
nism on limiting pantothenate, conditions will 
be found wherein a large proportion of the 
ACP of the organism will be apoACP. Conver- 
sion of apoACP to holoACP will be attempted 
with crude extracts of wild type E. coli. 



Additional Roles for Pantothenate-containing 
Proteins 

4'-Phosphopantetheine is the prosthetic 
group of at least one protein, ACP, of E. coli. 
Availability of E. coli mutants that require 
pantothenic acid for growth permitted the ad- 
ministration of C"-pantothenate to growing 
cells in order to establish the fate of this com- 
pound. The cells were then opened and fraction- 
ated into a particulate fraction and a soluble 
fraction. The proteins of the soluble fraction 
were separated from CoA and other non- 
protein components by filtration on Sephadex. 
Then the proteins were further fractionated by 
DEAE column chromatography. At least two 
protein peaks contained radioactivity derived 
from the C^^-pantothenate. In addition, the 
particulate fraction contained radioactivity. 
Experiments such as these promise to demon- 
strate the overall significance of protein-bound 
pantothenate, whether this be in the elucida- 
tion of other proteins with pantothenate 
prosthetic groups or in the possible implication 
of ACP-like proteins in such processes as 
cellular transport and permeation. The latter 
possibility has arisen with the finding that pro- 
tein-bound C"-pantothenate is found in the 
particulate ( ? membrane) fraction of the cell. 

Attempts to Demonstrate ACP in Mammalian 
Tissues 

The demonstration of ACP in bacteria has 
stimulated a search for a similar protein in 
other species. Recently P. Stumpf (University 
of California) reported partial purification of a 
similar protein from plants. Both bacterial and 
plant enzyme preparations for fatty acid 
synthesis are soluble, and thus isolation of indi- 
vidual native protein components is relatively 
simple. Fatty acid synthetase preparations 
from yeast or mammals are protein complexes, 
and fractionation of these complexes to yield 
active enzyme components or ACP has not yet 
been achieved. However, the demonstration of 
4'phosphopantetheine as the prosthetic group 
of E. coli ACP suggested a new approach to the 
question of whether the mammalian fatty acid 
synthetase preparation contains ACP. With the 
availability of a microbiological assay for pan- 
tothenate (which is derived from phosphopan- 



NATIONAL HEART INSTITUTE 



79 



tetheine as detailed above), purification of the 
rat enzyme which catalyzes fatty acid synthesis 
could be correlated with the pantothenate con- 
tent of the enzyme preparation. It was found 
that pantothenate copurifies with the fatty acid 
synthesis activity. In addition, C-'*-pantothen- 
ate was fed to pantothenate deficient rats, and 
the radioactive component was isolated from 
the purified enzyme system. This labeled com- 
ponent was shown to be 4'-phosphopantetheine 
by column chromatography and by enzymatic 
conversion to CoA. It is concluded that the 
mammalian fatty acid synthetase contains pro- 
tein-bound 4'-phosphopantetheine. Demonstra- 
tion of the entire mammalian ACP awaits 
further experiments. 

Section on Cellular Physiology 

The Cellular Physiology Section, in pursuing 
its program directed at the structural basis of 
the biochemical activities of proteins, their bio- 
synthesis and functional relationships in the 
integrated activity of cellular structure, is 
broadly divided into three main projects : 

(1) Studies on protein structure. These 
are primarily focused on the fibrous 
proteins involved in muscular con- 
traction and blood clotting, with ma- 
jor concern for substructure, and re- 
lationship of primary structure to the 
Configurational requirements of bio- 
chemically significant aggregation pro- 
cesses and enzyme activity. 

(2) Protein biosynthesis. Investigations 
in this area are concerned with the 
role of cellular membranes, the bio- 
synthetic unit and energetics. 

(3) The metabolic relationships of unsat- 
urated fatty acids. Investigations in 
this program are concerned with the 
metabolism, synthesis and function of 
unsaturated fatty acids in liver and a 
number of nucleated unicellular or- 
ganisms. 

Muscle Proteins: Myosin 

Previous reports have summarized investiga- 
tions leading to a model for the molecule as a 
long three-stranded rope with a globule at one 
end and composed of three physically and chem- 
ically identical polypeptide chains. Other in- 



vestigations have located the enzymic sites of 
the molecule in the globular portion and the 
amino terminal ends of the chains were tenta- 
tively identified as histidine and localized in this 
portion of the structure as well. Previous iden- 
tification of N-terminal histidine was based on 
an analytical procedure not entirely specific for 
histidine. Current activities have verified that 
amino terminal histidine is indeed recovered 
from myosin and its quantitation for the whole 
molecule and the globular end is in good agree- 
ment with the molecular weight of the subunit 
chains. 

Previously, methods have been developed for 
selectively labelling with radioactive sulfhydryl 
reagents each of the two cysteine residues asso- 
ciated with the active sites of myosin. Enzymic 
digestion and chromatographic fractionation 
and purification of the resulting peptide frag- 
ments have led to identification of a heptapep- 
tide involving one of the two cysteines and hav- 
ing the sequence: glutamyl glycyl isoleucyl 
arginyl isoleucyl cysteinyl arginine. The second 
cysteine residue is present in a tryptic nona- 
peptide with the composition and partial se- 
quence: cysteinyl aspartyl glutaminyl (glycyl, 
glycyl, valyl, isoleucyl, leucyl) arginine, the se- 
quence of the portion in parenthesis not having 
been established. 

Furthermore, most of the cysteine contain- 
ing peptides resulting from tryptic digestion of 
C"-iodoacetate labelled myosin have now been 
isolated and at least partial sequences estab- 
lished for the majority of them. Successful 
completion of this portion of the project in the 
very near future should provide final proof for 
the chemical identity of the polypeptide chains, 
at present based on three less direct lines of 
evidence. 

Bovine Fibrinogen 

Earlier studies established that fibrinogen 
has the same molecular weight both in the pres- 
ence of guanidinium chloride, which destroys 
the noncovalent bonding in proteins, and in di- 
lute salt solutions. However, it is well recog- 
nized from chemical studies that fibrinogen 
possesses 6 polypeptide chains. Determination 
of the molecular weight in guanidinium chlo- 
ride following cleavage of the disulfide bonds 
indicated that the molecule was composed of 



80 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



six polypeptide chains of essentially identical 
mass and indicating that the six chains of 
fibrinogen are all associated through interrelat- 
ed disulfide bridges. 

Analysis of the cleavage of fibrinogen by 
trypsin indicated that short digestion led to 
formation of three fragments of equal size 
which appears to result from the cleavage of 
the three beaded structure seen in electron mi- 
croscopy. Examination of the loss of clotting 
ability during the liberation of these three 
fragments indicated that loss of clotting ability 
occurs much faster than the cleavage reaction. 
However, this relationship is complicated by 
the fact that trypsin will split the thrombin- 
specific bonds in fibrinogen. Turning to chymo- 
trypsin with its very different peptide bond 
specificity, this enzyme also yields the same 
overall picture of digestion with initial release 
of three large fragments. In contrast to trypsin, 
the loss of clotting ability parallels the release 
of the fragments. Following fragmentation and 
loss of clotting ability, it was shown that 
thrombin will still split four peptide bonds per 
original fibrinogen molecule — the same as with 
native fibrinogen — indicating that fragmenta- 
tion has not altered the subtle specificity re- 
quirements of thrombin. 

Ribonuclease 

Previous work on the reaction of the fluores- 
cent dye 5-dimethylamino-l-naphthalensulfonyl 
chloride (DNS chloride) with ribonuclease has 
demonstrated that though the dye does not in- 
hibit the enzyme, it interferes with the reforma- 
tion of the native, enzymically active structure 
in reduction and reoxidation of the disulfide 
bridges. From proteolytic digestion and isola- 
tion of the DNS peptides, it seemed probable 
that this effect could be related to combination 
of the dye with certain specific histidine and/ 
or lysine residues. Owing to the lability of the 
bound dye, some ambiguities existed as to the 
site of attachment in these peptides. These 
have now been overcome and the DNS deriva- 
tive of histidine 48 — the major point of reac- 
tion — has been positively identified. 

In other investigations involving ribonu- 
clease, this protein has been used as a model to 
study the effects of gamma radiation on the 
structure and biological activity of proteins. 



These studies necessitated the development of a 
new technique for studying free radical forma- 
tion — in which they are trapped by reaction 
with tritiated hydrogen sulfide. The affected 
amino acid residues are then identifiable « 
through their radioactivity. Preliminary find- if 
ings indicate that with native ribonuclease, 
free radical formation occurs with lysine, j 
methionine, proline and histidine residues in % 
decreasing order. On the other hand, structur- 
ally altered, inactive ribonuclease shows an 
entirely different pattern of free radical for- 
mation. Studies on enzyme activity as a func- 
tion of irradiation suggest that structural 
changes are produced that leave active but al- 
tered forms of the enzyme. 

Protein Biosynthesis 

Previous reports have demonstrated the high 
metabolic activity of the lipid soluble amino 
acid and peptide fractions of hen oviduct and 
preliminary chemical characterization indicat- 
ed they were combined in the lipid structure 
through both amino and carboxyl ends. Though 
their metabolic activity made these lipopeptide 
materials eligible as intermediates in protein 
biosynthesis, no direct evidence for this exist- 
ed. Approaching the problem of obtaining pep- 
tide fragments from the lipopeptides for com- 
parison with fragments of ovalbumin obtained 
by the same fragmentation procedures, it was 
immediately evident that the lipopeptide ma- 
terial was rather refractory to standard hydro- 
lysis techniques. However, recent modifications 
have made possible a comparison of partial hy- 
drolysates of the two materials by the "finger- 
print" technique and have indicated, within the 
limitations of this method, that several of the 
characteristic peptides of ovalbumin may be 
present in the lipopeptide material. 

Nonphosphorylated High Energy Interme- 
diates and Protein Biosynthesis 

Recent work in a number of laboratories has 
indicated that the electron transport system of 
mitochondria gives rise to high energy in- 
termediates which may be available to ender- 
gonic processes such as reversal of electron 
transport, ion accumulation and amino acid up- 
take prior to their involvement of phosphate 
and ATP formation. In the present work, pro- 



NATIONAL HEART INSTITUTE 



81 



tein synthesis by anaerobically growing yeast 
was inhibited by the classical uncouplers of ox- 
idative phosphorylation, dinitrophenol and 
azide. These agents do not inhibit anaerobic 
glycolysis and ATP levels were the same as in 
the uninhibited controls. Insofar as can be 
detected with current methodology, there is no 
interference by these agents in the maintain- 
ance of amino acid and nucleotide pools. On the 
other hand, neither dinitrophenol nor azide in- 
hibits the classical in vitro system for incorpo- 
ration of amino acids into ribosomes. 

Synthesis of Long-Chain Unsaturated Fatty 
Acids 

Previous work with guinea pig liver enzyme 
preparations has demonstrated the formation 
and interconversion of long chain «,/? and /3,y- 
unsaturated and /^-hydroxy fatty acids. It has 
now been shown that guinea pig liver mito- 
chondria contain two hydrases which catalyze 
the hydration of ira.?is-a,j8-hexadecenoyl-CoA to 
D- ( — ) -jS-hydroxypalmityl-Co A. No evidence 
could be found for the hydration of the I3,y- 
CoA derivative. An enzyme preparation has 
been obtained that catalyzes the direct reduc- 
tion of the cis- and trans-fS,y- and trans-a,l3-hex- 
adecenoyl-CoA compounds. All of the following 
reactions have now been demonstrated using 
partially purified enzymes. 

palmityl-CoA 

/ ^ 

«rans-a^-hexadecenoyI-CoA=r irons- and cis-ft7-hexadecenoyl-CoA 

IT 

D-(— )-<S-hydroxypalmityI-CoA 

In a study of the synthesis of polyunsaturated 
acids, it has been shown that a species of soil 
amoeba, Acathamoeba, normally contains poly- 
unsaturated fatty acids with the last double 
bond 6 carbon atoms removed from the methyl 
end of the fatty acid. When grown in the pres- 
ence of linolenic acid, a Cis triunsaturated acid 
with the last double bond 3 carbon atoms re- 
moved from the methyl end, the organism con- 
verts it to the corresponding C20 tri, tetra and 
penta unsaturated acids suggesting that the en- 
zyme systems for chain elongation and intro- 
duction of additional double bonds present in 
these organisms are sufficiently non-specific to 
be uninfluenced by the presence of a double 



bond 3 carbon atoms closer to the methyl ter- 
minus. 

In an examination of the unsaturated fatty 
acid metabolism of Tetrahymena, it has been 
demonstrated that this organism normally con- 
tains only 2 polyunsaturated fatty acids, lino- 
leic and y-linolenic acids. However, when the 
organisms are fed a number of unsaturated 
acids, many are converted to a variety of unnat- 
ural polyunsaturated acids. Some of the unnat- 
ural unsaturated acids added to the growth 
medium and some of the unnatural acids 
formed are incorporated into the phospholipids 
of the organism — an observation that has some 
implications as to the function of polyunsatu- 
rated fatty acids. It is strange that y-linolenic 
acid which normally accounts for 60% of the 
fatty acids of the organism is lethal when 
added to the medium. 

The Biochemistry and Cytology of Cell Trans- 
port 

Previous studies on the active uptake of ra- 
dioactive fatty acids by the soil amoeba, 
Acanthamoeba, demonstrated that most of the 
fatty acid was initially present in the cell in 
the form of unesterified acid, suggesting that 
active uptake may not involve an esterification 
step. The metabolic fate of the C"-palmitate 
has now been followed and the kinetics of its 
conversion to phospholipids and glycerides de- 
termined. The esterified forms appear to be 
largely membrane bound. The phospholipid frac- 
tion has been fractionated and is present as 
phosphatidyl choline, phosphatidyl serine and 
phosphatidyl ethanolamine. The fatty acid 
composition of each fraction has been complete- 
ly analyzed. 

Together with the preliminary application of 
electron microscopy to the cytological aspects 
of this process, the chemistry of some of the 
staining and fixation procedures commonly em- 
ployed in electron microscopy have been exam- 
ined. It appears that osmic acid reacts with 
the unsaturated fatty acyl groups to form the 
osmic acid esters of the dihydroxy derivatives 
and not with the polar portion of the phospho- 
lipids which is contrary to current concepts on 
which present views of the molecular orienta- 
tion in cell membranes is based. 



82 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



The results of studies on the stability of lip- 
ids after various fixing procedures should 
have wide implications in the interpretation of 
electron micrographs. Thus, it has been deter- 
mined that after glutaraldehyde fixation, the 
subsequent dehydrating steps remove all of the 
lipids from the tissues. With KMnO* fixation, 
all of the neutral lipid and some of the phos- 
pholipid is removed. When OSO4 is used, some 
of the neutral lipid and none of the phospholip- 
id is removed. 

LABORATORY OF CHEMICAL PHARMA- 
COLOGY 

Biological Control Systems 

Adrenergic Neurochemical Transducer 

In the last report a theoretical model of the 
control system at sympathetic nerve endings 
which integrates processes that synthesize, 
store, inactivate and release NE was described. 
It was termed a "neurochemical transducer" 
since it translates electrical impulses into a 
precise quantity of free neurohormone, which 
in turn acts on a target organ and releases me- 
chanical or physical energy. The original struc- 
ture for the adrenergic transducer, formulated 
by tracing out various components of the 
amine store with highly labeled H^NE has 
stood up well. Its predictive value in expanding 
our knowledge of nerve function and of drug 
action has been great. 

The following is the postulated model for the 
NE neurochemical transducer: NE is separated 
from receptors and monoamine oxidase 
(MAO) by a lipid membrane. The amine is pres- 
ent in a mobile pool which is in chemical equi- 
librium with a reserve pool, probably a com- 
plex of NE in granules. NE in the mobile pool 
is maintained within the membrane by a spe- 
cialized concentrating system or NE pump. 
The constant concentration of NE in nerve 
endings results from a balance of continuous 
synthesis and loss of amine. MAO in mitochon- 
dria inactivates the NE that constantly diffuses 
from sites of storage so that the steady-state 
level is below that which will saturate the trans- 
port system. Nerve impulses depolarize the spe- 
cialized membrane facing the receptor there- 
by altering the permeability to NE and releas- 



ing amine to receptor sites. Between each im- 
pulse, the membrane permeability is restored 
and NE released onto the receptor is recap- 
tured by the nerve ending by the action of the 
pump. The uptake of NE was postulated to be 
highly stereospecific for the 1-isomer; we have 
now shown this to be true. In addition, the 
sympathetic nerve endings retain E as well as 
NE but do not retain 5HT or histamine. 

Release of NE by Various Drugs 

In the proposed model, NE diffuses contin- 
uously from the mobile pool onto MAO at a 
rate proportional to concentration. Reserpine 
is postulated to block the NE pump, thereby in- 
creasing the rate of net efflux onto MAO. In 
contrast, nerve impulses and a number of 
drugs such as tyramine and guanethidine, are 
thought to depolarize the membrane directly in 
front of the receptor thereby releasing NE 
onto receptor sites. Other workers, however, 
hold that these drugs act on the granules them- 
selves. 

After NE synthesis is blocked by a-methylty- 
rosine, the concentration of amine in brain and 
heart declines exponentially. These results dem- 
onstrate that the passage of catecholamine 
from the complex in granules to the mobile 
pool can be explained by simple mass action 
kinetics. 

Reserpine depletes NE by increasing its rate 
of efflux in the mobile pool; the loss of NE is 
still proportional to the concentration but the 
rate constant is increased. From the kinetics of 
NE loss, the degree to which reserpine inhibits 
the NE pump may be calculated. 

Reserpine appears to act by removing a re- 
sistance (the NE pump) to the free flow of the 
monoamine from nerve endings. Accordingly 
by relating the NE efflux in control rats to the 
NE efflux when the amine is released at a max- 
imal rate, the activity of the NE pump can be 
calculated. The results are consistent with the 
view that reserpine acts on the membrane 
transport rather than on the granule complex. 

Tyi-amine, infused so as to produce a high 
plasma level, also depletes heart NE by in- 
creasing its efflux from the mobile pool. The 
loss of NE, which is released onto receptor 
sites as the free base, is exponential with no 



NATIONAL HEART INSTITUTE 



83 



evidence of a tyramine-resistant pool or of ta- 
chyphylaxis. These results indicate that the 
dissociation of the NE complex to readily avail- 
able amine is not the rate-limiting step. The 
maximal rate of release (ti^z = 50 minutes) is 
about one-third that after reserpine, suggest- 
ing that the NE pump has not been blocked but 
that the amine is released only from the mem- 
brane opposite the receptor, whereas after res- 
erpine it is released over a larger membrane 
surface. Metaraminol and octopamine, in vivo, 
and guanethidine in vitro also release NE onto 
receptors as the base at the same maximal rate 
as tyramine, indicating that these drugs all re- 
lease the amine by a similar mechanism. 

The effects of MAO inhibition support the 
view that tyramine does not act directly on 
granules. Thus blockade of MAO slows the rate 
at which NE is released by reserpine, but does 
not aif ect that produced by tyramine. 

After depletion of heart NE by tyramine, the 
stores of NE can be replenished by the injec- 
tion of exogenous NE, showing that tyramine, 
and presumably guanethidine and aramine, do 
not affect the intrinsic storage mechanism. 

Mechanisvis of NE Release at Molecular and 
Subcellular Levels 

The distribution of metaraminol in vivo is 
indicative that the drug is an almost ideal tool 
for studies of the mechanism that stores and 
releases NE. It is taken up only by adrenergic 
neurons and it is not bound to non-specific 
sites. Preliminary results suggest that the drug 
is distributed in almost exact proportion to the 
concentration of endogenous NE in tissues ; the 
use of the labeled drug might therefore provide 
a simple way to compare NE levels in various 
tissues. Preliminary results indicate that meta- 
raminol is readily taken up by nerve endings of 
rats given reserpine. However the amine disap- 
pears at a relatively rapid rate since the sys- 
tem is now open-ended. 

Metaraminol was found to be localized in 
heart granules of rats pretreated with des- 
methylimipramine. Since this drug blocks the 
release of NE elicited by metaraminol, it is un- 
likely that the latter drug releases NE by a di- 
rect action on granules. 



Heart slices, incubated in vitro, accumulate 
metaraminol by a specialized transport system. 
In contrast, the drug is taken up by skeletal 
muscle by passive diffusion. Since the steady- 
state level achieved by both processes is 
reached at the same time, the storage process 
may be regarded as a pump and leak process. 
The uptake process is saturated when the con- 
centration of metaraminol inside heart tissue 
reaches 0.7 microgm/g. This level compares 
with the 1.1 microgm/g of NE normally pres- 
ent. The accumulation of drug and the release 
of NE appear to be separate processes ; the up- 
take of metaraminol is relatively rapid while 
the release of NE is much slower. The extent 
of NE depletion is related not only to the level 
of metaraminol but also depends on the incuba- 
tion time. 

Heart slices from rats treated with reserpine 
take up metaraminol at the same rate as would 
be predicted from the rate constant for passive 
diffusion but the diffusion process continues 
over a long period of time until the final con- 
centration of drug in tissue becomes equal to 
that in control slices. Skeletal muscle slices do 
not exhibit similar behavior. According to our 
model, these results would be explained by a 
slow passive diffusion of the drug together 
with its reaction with granules to form a com- 
plex of high affinity. Substantiation of this 
view would be definite proof that reserpine 
does not act directly on granules. 

To achieve similar conditions with NE, heart 
slices taken from animals treated with reser- 
pine and a MAO inhibitor (to protect the NE) 
are incubated with H^NE. The uptake of 
H^'NE at equilibrium is 2.5 times that in tis- 
sues of animals that received only reserpine. 
These results indicate that reserpine blocks ac- 
tive transport at the neuronal membrane but 
does not act directly on granules. 

The Effects of Desmethylimipr amine (DMI) 
on Uptake and Release 

DMI is proving to be a pharmacological tool 
of considerable importance in unravelling the 
nature of the adrenergic neuron. DMI, which by 
itself exerts no pharmacological effects, rev- 
erses reserpine depression to a pronounced ex- 
citation. This prompted us to study the effects 



84 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



of DMI on the uptake and release of NE at pe- 
ripheral adrenergic neurons. At first these 
effects seemed paradoxical, since DMI blocks 
the uptake of H^NE given in tracer doses 
without releasing endogenous NE and without 
apparently blocking the uptake of large doses 
of NE. DMI exerts similar effects on drugs 
which are stored by adrenergic neurons; for 
example it prevents the uptake of guanethi- 
dine, metaraminol and tyramine given in small 
doses, but does not release these drugs after 
they have been taken up by nerve endings. 
Moreover, DMI does not block the uptake of 
these drugs when they have been given in large 
doses, but invariably prevents them from re- 
leasing NE. 

The finding that a given dose of DMI blocks 
the uptake of H^NE by the same proportion 
over a considerable dosage range suggested 
that DMI might decrease the intrinsic permea- 
bility of the neuronal membrane to NE. Thus, 
20 mg/kg DMI blocks H^NE uptake by 95% 
and would be said to block almost completely 
the uptake of tracer doses of the catechola- 
mine. But the inability of DMI to block the up- 
take of large doses of NE and of various drugs 
stored by the adrenergic neuron is only appar- 
ent. The small proportion of NE or drug up- 
take is now sufficient to saturate the binding 
capacity of the adrenergic neuron. 

Although DMI does not prevent the uptake 
of NE after large doses of the amine, it pre- 
vents this NE from displacing H^NE already 
in the nerve endings. Thus DMI inhibits the 
passage of NE out of, as well as into, the nerve 
endings. Moreover it seems to separate the 
granule from the mobile pool. In support of the 
view that DMI affects membrane both at the 
neuronal membrane and at granules, the drug 
decreases the spontaneous loss (turnover) of 
H^NE from rat heart. In addition it decreases 
the quantity of NE released by nerve impulses 
though it also potentiates the biological re- 
sponse. 

The Recapture Process 

The inability of nerve stimulation to deplete 
NE stores, despite a slow turnover of the amine, 
may be explained by the model. Between 
successive nerve impulses, the impermea- 



bility of the neuronal membrane is re-estab- 
lished and amine released to receptors will be 
pumped back into nerve endings and used 
again. Since phenoxybenzamine prevents this 
re-uptake, this drug provides a good tool for a 
study of the recapture mechanism. In the cat- 
colon preparation, this drug increases about 
5-fold the H^NE output produced by electrical 
stimulation. It is generally believed that phen- 
oxybenzamine blocks directly the NE uptake by 
nerve endings. Our results show that phenoxy- 
benzamine, after it has acted irreversibly, 
blocks the uptake of H^NE to only a minor de- 
gree. The drug prevents the attachment of NE 
to the receptor site and allows it to escape into 
the blood stream while the nerve terminal is 
still depolarized. In contrast, cocaine (and 
DMI) actually blocks the uptake of NE and in 
fact lowers the output of NE by nerve stimula- 
tion. Further evidence that the two drugs act 
differently is the finding that phenoxybenza- 
mine increases the spontaneous outflow of 
H^NE (normal tone) while cocaine diminishes 
it. Thus phenoxybenzamine acts by blockade of 
receptors, while cocaine and DMI act by block- 
ade of the uptake. This particular action of 
adrenergic blocking agents should be impor- 
tant in differentiating alpha and beta blocking 
agents in biochemical terms. 

The difference between the output of H^NE 
after sympathetic stimulation of control and 
phenoxybenzamine-treatd colon, has permitted 
the calculation of NE released per nerve im- 
pulse and of the extent to which the recaptured 
mechanism preserves the amine supply. Such 
calculations show that nerve endings would 
soon be depleted of their neurohormone if they 
lacked the reuptake process. 

Rates of NE Synthesis 

The rate of formation of NE may be calcu- 
lated from rate of NE los after blockade of 
synthesis by a-methyltyrosine 

[NE] = [NEo]e->^' 

where NEo is the initial level of NE 



In 



[NE] 
[NEo] 



= -kt 



NATIONAL HEART INSTITUTE 



85 



[NE] 

A plot of In vs time yields the fractional 

[NEo] 

turnover rate constant and k [NEo] = synthesis 

rate. 

Synthesis rate in heart = 0.05 microgm/g/hr 

Synthesis rate in brain = 0.12 microgm/g/hr 

The rate of formation of NE in heart may also 
be calculated from the rate of refilling of the 
NE stores after depletion by tyramine. 

[NE] = [NEo] (1 - e-") 

where NEo is the final level of NE 

InH^.kt 
[NE„] 

[NE] 
A plot of In vs time yields fractional 

[NEo] 

turnover rate constant and 

k [NEo] = synthesis rate 

= 0.05 microgm/g/hr 

Role of Inorganic Ions in Storage and Release 
of NE 

Because of the importance of electrolytes in 
depolarization phenomena and in biological 
processes, we are now studying the effects of 
electrolyte changes on the storage and release 
of NE in heart slices taken from rats injected 
with tPNE. The rapid efflux of radioactivity 
that occurs when the slices are first incubated 
with Krebs-Ringer solution, appears to result 
from depolarization and soon subsides. The 
rate of efflux of H'NE now becomes slow and 
declines exponentially. 

Depolarization elicited by increasing exter- 
nal K+ enhances the rate of H^NE release; at 
maximum depolarization, the rate constant of 
release is identical to that elicited by guanethi- 
dine, suggesting that the drug also releases NE 
membrane depolarization. Addition of bretyl- 
ium to the incubation fluid blocks the release 
of NE elicited by guanethidine. 

In the absence of external Ca, the efflux of 
NE depends on the ratio of K+ to Na+ and 
maximum retention of the amine is obtained in 
a ratio of about 10 to 1. This suggests that an 



ATPase is involved in maintaining the NE 
pump. 

The most rapid efflux of NE occurs in the 
complete absence of Na+, suggesting that the 
uptake process might be linked to the Na* 
pump. It is possible that the rate of effiax in 
this case is the same rate as that produced by 
reserpine. 

Ouabain blocks the uptake of NE by heart 
slices; in addition it releases NE from heart 
slices in a Ca free medium. 

Other Research Items 

Release op Drugs by Nerve Stimula- 
tion. We have continued our studies of drugs, 
such as tyramine and guanethidine, which are 
taken up by adrenergic neurons and then ap- 
pear to elicit persistent depolarization. In sup- 
port of the view that they are stored in sym- 
pathetic nerve endings, our results show that 
they are released by electrical stimulation. In 
addition, reserpine interferes with their up- 
take. 

The Subcellular Action of Drugs. The 
presence of NE in granules (vesicles) and the 
selective action of various drugs makes it pos- 
sible to study drug action at a subcellular level. 
For example, bretylium, DMI and certain MAO 
inhibitors prevent guanethidine, tyramine or 
metaraminol in pharmacological doses from re- 
leasing NE but do not block the uptake of the 
latter drugs by adrenergic neurons. It may be 
presumed that the release of NE is prevented 
by an action within the neuron itself. 

Bretylium or DMI might conceivably block 
depolarization elicited by guanethidine or 
metaraminol, or prevent these substances from 
displacing NE from granules. Since neither 
DMI nor bretylium prevents the uptake of 
metaraminol by granules, it is unlikely NE is 
released by metaraminol in this way. 

However, the techniques for isolating gran- 
ules have given such inconsistent results that 
this approach has been suspended in order to 
examine the effects of tissue fractionation on 
granule NE. Preliminary results suggest that 
the leakage of NE from synaptic vesicles, 
rather than breakage of vesicles, might be the 
main reason for the loss of NE. Steps are un- 
derway to remedy this loss. 



86 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Use of Drugs in Measurements of NE 
Compartments. Results from this laboratory 
have shown that the amine compartments can 
be traced out by means of H^NE administered 
in tracer doses. Conclusions about amine com- 
partments are difficult to make if nonsteady 
conditions prevail. Thus, based on release of 
NE by reserpine and tyramine in large doses, 
it might be concluded that NE is present in a 
single compartment, since both pools are in 
dynamic equilibrium. Again the ratio of the 
efflux of H^NE relative to the efflux of endo- 
genous NE will not disclose information on 
pool size but will simply show conditions under 
which passage from one pool to another, or the 
effect of the disappearance of drug itself, be- 
comes rate limiting. 

Bretylium. Although bretylium, like 
guanethidine, prevents the nerve impulse from 
releasing NE, it apparently acts by a different 
mechanism as described in previous reports. 
Our studies now show that reserpine releases 
guanethidine but not bretylium; catechola- 
mines can compete for the uptake of H^-guan- 
ethidine but not for that of C"-bretylium ; 
pretreatment with large doses of guanethidine 
dilutes out the uptake of H^'-guanethidine, but 
large doses of bretylium do not dilute out the 
uptake of H^-bretylium. Thus the two drugs 
are incorporated by different neuronal sites. 

An Indirect Effect of Reserpine on 
Granules. After depletion of heart NE by the 
injection of reserpine, amine levels are com- 
pletely depleted for 2 to 3 days. In about 20 
hours, however, the nerve endings recover in 
part the ability to take up H^NE but not to store 
the amine. These results indicate that the NE 
pump recovers before the granules are able to 
form a stable complex with the amine, and sug- 
gest that one of the complexing components 
has disappeared. 

The Serotonergic Transducer 

A definite role has not yet been assigned to 
5HT in brain because of the inaccessibility of 
brain neurons and the difficulty of interpreting 
the effects of endogenous 5HT, e.g., small doses 
of 5HTP elicit sedation while large doses elicit 
excitation. Although 5HT and NE transducers 



can be described in almost identical terms, few 
drugs seem to have a specific action on the 5HT 
transducer. Thus no substance has been found 
that mimics, blocks the action of, or blocks the 
synthesis of 5HT. In fact the main clue for the 
action of 5HT has been reserpine, which blocks 
the storage of both 5HT and the catechola- 
mines. If it could be established that sedation 
were attributable mainly to changes in 5HT, 
reserpine would assume an important role in 
studies of the physiological function of this 
amine. 

The catecholamine amines are now definitely 
eliminated from the picture of reserpine seda- 
tion, since a-methyltyrosine, which specifically 
blocks the formation of catecholamines, elicits 
no sedation in doses that deplete the amines by 
80%, and produces typical chlorpromazine-like 
effects when depletion is 90% or more. Last 
year we reported that recovery from reserpine 
was closely related with the recovery of brain 
tissue's capacity to take up 5HT formed from 
exogenous 5HTP. That reserpine acts through 
constant occupancy of receptors by 5HT is now 
virtually proved. First, excitation elicited by 
large doses of 5HTP is not due to the action of 
5HT at serotonergic receptor sites but to dis- 
placement of brain NE by 5HT formed in 
adrenergic neurons. Second, a high concentra- 
tion of free 5HT (62 ng/g) exists at nerve end- 
ings after the complete depletion of amine 
stores. Most importantly, after blockade of 
catecholamine amine synthesis, a MAO inhibi- 
tor given to rats treated with threshold doses 
of reserpine, elicits a profound sedation that is 
correlated with the rise in brain 5HT. 

Reserpine no longer produces tremors in ani- 
mals when catecholamines synthesis has been 
blocked. These results suggest that the tremors 
arise from persistent occupancy of dopamine 
receptors in caudate nucleus. This would ex- 
plain why reserpine tremors are blocked by 
chlorpromazine but not by atropine. 

Effects of Drugs in Various Phyla 

The brain of the lamprey, the most primitive 
of fish, contains 5IIT and dopamine, but no NE 
or E. Only in higher classes of fish does NE 
appear in brain. Amphibian brain contains E. 
but no NE. The E, however, is resistant to re- 



NATIONAL HEART INSTITUTE 



87 



lease by reserpine or any other drug we have 
tried. 

The action of reserpine in lower animals is 
dependent upon the rate of 5HT formation. In 
mammals and birds for example, reserpine al- 
ways produces sedation and 5HT is formed at a 
high rate. In amphibia, the formation of 5HT 
is slow; although reserpine releases 5HT 
stores, it does not elicit sedation unless the ani- 
mals are also given a MAO inhibitor to protect 
the liberated amine. 

In frogs, the administration of a MAO inhib- 
itor produces sedation after a period of 24 
hours or longer. At this time there is no rise in 
brain E, but 5HT levels have risen and have 
reached a peak. The following drugs elicit se- 
dation in frogs pretreated with MAO inhibi- 
tors: a-methyl-metatyrosine, dopamine, 5HTP, 
amphetamine. None of these drugs elicits seda- 
tion by itself and in each case the sedation is 
related to the presence of a pool of free 5HT. 

Nonmast Cell Histamine 

Last year we reported that highly labeled 
H^-histamine injected in tracer amounts selec- 
tively labeled the nonmast cell histamine 
stores. Such histamine was found to be present 
in all tissues and is not released by compound 
48/80. The assay of H'-histamine by isotopic 
dilution now makes it evident that the reported 
diphasic disappearance of the labeled hista- 
mine is true only in part. The second compo- 
nent (half -life of 150 hours) is not that of hista- 
mine but that of a metabolite of unknown 
structure, which is distributed evenly in body 
water and is poorly excreted. The kinetics of 
distribution of H^-histamine indicates that 
nonmast cell histamine has a rapid turnover, 
about 2 hours. 

Contrary to the general impression, some an- 
imal species (cat and rabbit) possess relatively 
little mast cell histamine and large amounts of 
nonmast cell amine. For example, except for 
the skin, the tissues in cats and rabbits contain 
mainly nonmast cell histamine. 

The following results, obtained mainly by ex- 
periments on the submaxillary gland, suggest 
that the action of the parasympathetic nervous 
system on exocrine glands may be mediated by 
nonmast cell histamine : 

H^-histamine in cat submaxillary gland de- 



clines with a half-life of 2 hours. Stimulation 
of the chorda tympani for 3 hours releases con- 
siderable amounts of labeled histamine into sa- 
liva as histamine metabolites; the H^-hista- 
mine concentration in the gland is reduced by 
about 80% relative to the unstimulated gland. 
Changes in the rate of salivation, produced by 
changes in frequency or voltage of electrical 
stimulation, are closely correlated with changes 
in the amount of label in the saliva. 

This relationship was further established by 
studies showing that salivation and the release 
of labeled histamine are blocked by atropine, 
while they are enhanced by eserine. Other 
agents that enhance both phenomena are ace- 
tylcholine, tremorine, reserpine and philocar- 
pine. 

Pilocarpine appears to have more than one 
action. It acts like acetylcholine and in addi- 
tion it depletes nonmast cell histamine, at least 
in the submaxillary gland. Thus, pilocarpine 
is a valuable tool in studying histamine com- 
partments and in relating cholinergic effects to 
histamine content. For example, shortly after 
H^-histamine is given, pilocarpine releases a 
large proportion of H'-histamine and only a 
small proportion of the endogenous amine, sug- 
gesting that histamine is located in more than 
a single compartment. However, after 1 to 2 
hours, the labeled and endogenous amines are 
released to the same extent. Large doses of pi- 
locarpine release a large fraction of endogen- 
ous amine after which the gland is refractory 
to stimulation of the chorda tympani. Pilocar- 
pine also increases the flow of saliva, gastric 
juice, bile, pancreatic juice, tears, nasal secre- 
tion and sweat. Bile and pancreatic juice, 
which have been studied in detail, contain con- 
siderable amounts of labeled histamine metabo- 
lites. 

Infusion of the histamine analogue, Histalog, 
considered to be specific for secretion of gastric 
juice, also enhances secretion of all the exo- 
crine glands. Priscoline produces similar re- 
sponses and, in addition, elicits peripheral vas- 
odilation. This is of particular interest in view 
of our ignorance of the control of microcircu- 
lation. 

The polypeptide gastrin releases consider- 
able amounts of both labeled and endogenous 
histamine from the stomach, but not from 



88 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



other organs. This action is not blocked by 
atropine and suggests that gastric action may 
be mediated through histamine release. Secre- 
tin produces a profuse flow of pancreatic juice 
and bile which contain considerable amounts of 
labeled metabolites. 

Nonmast cell histamine accounts for the bulk 
of the amine formed in the body. It is not clear 
how the processes of synthesis, binding, release 
and metabolism of nonmast cell histamine are 
integrated, but the following picture is emerg- 
ing: at the steady state, nonmast cell histamine 
is formed and inactivated at a rapid rate, the 
metabolites diffusing into the blood stream. 
The synthesis and utilization of histamine are 
closely linked processes. Atropine slows down 
the normal turnover of labeled histamine, in 
addition it reduces the incorporation of 
H^-histamine from the H=-histidine into endo- 
genous stores in the gastric mucosa and sali- 
vary gland. These results suggest a feedback 
mechanism by which histamine inhibits histi- 
dine decarboxylase, thereby permitting a pre- 
cise adjustment of synthesis according to need. 
This view would explain why histamine stores 
are not depleted even after prolonged nerve 
stimulation. In support of this view, the turn- 
over of histamine in the gastric mucosa is re- 
duced to about 20% of normal, when the rat 
esophagus is tied off to prevent the entrance 
into the stomach of a salivary factor known 
to promote gastric secretion. 

Since H^-histamine is released into the exo- 
crine secretions as metabolites, histamine must 
be stored in the secreting cell. Moreover, ace- 
tylcholine from cholinergic nerve endings must 
also act on, and the metabolizing enzyme must 
occupy this cell. Since the usual inhibitors do 
not protect the histamine released by nerve 
stimulation, the enzyme inside the target cell 
is not one of those that inactivate circulating 
histamine. Gastrin and other polypeptide hor- 
mones of the GI tract release histamine by a 
different and more specific mechanism not 
inhibited by atropine. 

Sympathetic Target Sites 

Mammals are able to make constant adjust- 
ments to the environment by a unique kind of 
adaptation in which enzymes are activated al- 



most instantaneously by the nervous system. 
We are now studying three tissue systems 
which respond to sympathetic stimulation 
through enzyme activation and increased mobi- 
lization of energy substrates — adipose tissue, 
the liver and skeletal muscle. The cells of these 
organs are transducer systems in which the in- 
put is NE and the output FFA, glucose or glu- 
cose-phosphate. We are particularly concerned 
with the way in which a physiological signal — 
a catecholamine released to the target cell — is 
converted to a biochemical signal, e.g., cyclic 
AMP inside the cell. 

Earlier studies showed that the functionally 
sympathectomized rat (adrenal demedullation 
and loss of peripheral stores) is unable to mo- 
bilize energy substrates. Such animals rapidly 
die on exposure to cold and collapse after 
forced exercise. The fact that these animals re- 
spond like adrenalectomized (ADX) rats led to 
studies which demonstrated that the sympathe- 
tic nervous system of these animals was unable 
to respond to catecholamines. 

Adipose Tissue System 

Since catecholamines, ACTH, and glucagon 
can elicit the same maximum rate of lipolysis, 
it is generally assumed that the rate of this 
process is limited by the amount of lipase. The 
finding that this rate is increased by theophyl- 
line led us to study this drug in some detail. 

Incubation of theophylline with adipose tis- 
sue or with fat cells produces a lipolytic re- 
sponse (as measured by glycerol output) hav- 
ing a maximum 3 times that yielded with NE 
or E. Theophylline, in an amount that elicits no 
response by itself, potentiates the lipolysis in- 
duced by NE and ACTH, but the resulting activ- 
ity never exceeds the maximal response elicit- 
ed by theophylline itself. These results suggest 
that the activation of lipase by catecholamines 
is normally limited by the low steady-state lev- 
el of cyclic AMP and that theophylline, by 
blocking phosphodiesterase, causes cyclic AMP 
to accumulate to a concentration that produces 
a maximal and perhaps a complete activation 
of lipase. In direct support of this view, theo- 
phylline causes a remarkable accumulation of 
cyclic AMP, which is closely associated with 
the lipolytic response; at a high concentration 



NATIONAL HEART INSTITUTE 



89 



of theophylline, the cyclic AMP level is in- 
creased by 6 times. Of particular interest is the 
finding that the maximum effect of theophylline 
is diminished in high doses; a similar finding 
has been reported for the action of cyclic AMP 
on lipase. Since theophylline potentiates NE 
and ACTH to the same degree, ACTH as well 
as NE may act through cyclic AMP. 

Interaction of Sympathetic and Thyroid Sys- 
tems 
This action of theophylline makes it a val- 
uable tool in determining whether a physiolog- 
ical change in lipolytic activity is caused by a 
change in lipase or in the ability to activate 
the enzyme. The effects of theophylline on the 
adipose tissue of thyroidectomized rats indicate 
that the slow rate at which cyclic AMP is 
formed is responsible for the poor responsive- 
ness to catecholamines. In addition, the hyper- 
responsiveness of adipose tissue from hyper- 
thyroid rats results from the rapid rate at which 
eyelid AMP is formed. These results suggest 
that the interaction of the thyroid and sym- 
pathetic systems might involve induction of 
adenyl cyclase. In accord with this view, the ac- 
tivity of adenyl cyclase in rats is tripled after 
pretreatment of rats with thyroxine, and this 
activity is closely related to the lipolytic re- 
sponse to NE. 

Nicotinic Acid-Induced Inhibition of Ldpolysis 

In vitro studies show that nicotinic acid, un- 
like the adrenergic blockers, inhibits the re- 
sponse to theophylline much more readily than 
to NE. Inhibition occurs at concentrations of 
lO-'M and maximum inhibition at about 
10-^M; in higher concentrations the effects 
are diminished. The addition of theophylline 
reduces the action of nicotinic acid indicating 
competition between the two drugs. These re- 
sults suggest that nicotinic acid might activate 
phosphodiesterase. This view is supported by 
results which show that when nicotinic acid is 
added to an incubation medium containing 
phosphodiesterase and cyclic AMP, the hydrol- 
ysis of the latter is increased. 

Possible Mechanisms of Alpha and Beta 
Adrenergic Blockade 
The lipolytic action of low concentrations of 
dichloroisoproteranol (DCI) is barely measur- 



able when the latter is used alone, but is mark- 
edly potentiated by low concentrations of theo- 
phylline. 

In higher concentrations, both DCI and 
phentolamine inhibit the lipolytic effects of NE 
and theophylline. DCI acts competitively 
against NE and is much more effective in 
blocking the response to NE than that to theo- 
phylline. In contrast, phentolamine blocks the 
NE and theophylline effects to the same extent 
and acts noncompetitively against NE. These 
results suggest that DCI acts primarily at the 
receptor site and that phentolamine acts pri- 
marily at a step between the production of cyc- 
lic AMP and the activation of lipase — perhaps 
on the action of cyclic AMP itself. In very 
large doses, DCI also blocks the effects of theo- 
phylline but this effect may be secondary. 

Lipolytic Effects on Theophylline in Vivo 

Theophylline in vivo produces a marked in- 
crease in the plasma levels of both FFA and 
glucose, and together with NE elicits a more 
than additive increase in FFA. The maximum 
effect of theophylline alone or theophylline to- 
gether with NE is greater than the maximum 
response to NE alone. 

Of great potential importance is the finding 
that the maximal effects of theophylline in chem- 
ically sympathectomized rats, or on fat pads 
from these animals, are practically the same as 
in intact animals. This indicates that the sym- 
pathetic nervous system is not required for the 
action of theophylline. It may be concluded that 
cyclic AMP is continuously released irrespec- 
tive of the nervous system. It is possible that 
changes in this basal turnover might explain 
the changes in lipolytic activity that occur in 
metabolic states like starvation. 

Biochemical Lesions in Adrenalectomy 

Epinephrine and glucagon increase the 
plasma glucose level in normal but not in ADX 
animals. ADX animals maintained for a couple 
of days on normal saline respond to epineph- 
rine and glucagon with a moderate increase in 
blood glucose, but 3 hours after pretreatment 
with a glucocorticoid, respond normally. Liver 
phosphorylase activity also increases after 
epinephrine or glucagon treatment in normal 



90 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



animals but in ADX animals, maintained on 
water, the increase in phosphorylase is far less 
significant. Pretreatment with a glucocorticoid 
restores the response to that in normal ani- 
mals. 

Cyclic AMP level in liver is increased about 
2-fold one minute after epinephrine or gluca- 
gon injection in normal animals but is not in- 
creased in ADX animals. After pretreatment 
with corticoids the response is partly restored. 

Cyclic AMP does not penetrate cells in 
appreciable amounts except after large doses. 
Five minutes after injection of 10 mg/kg of 
cyclic AMP, the concentration of label in liver 
is about 5 times that in muscle and 20 times 
that in blood and a moderate increase in blood 
glucose is elicited. The effect is less dramatic 
and quite variable with ADX animals. Cyclic 
AMP activates liver phosphorylase to the same 
extent in normal and in ADX animals main- 
tained on water. 

Thyse results suggest that the biochemical 
lesion in ADX animals is centered around cyc- 
lic AMP. The reactivity of animals to epineph- 
Tine might depend on an optimum ionic envi- 
ronment which is lacking in ADX animals and 
is restored by corticoids. 

In normal and ADX animals, theophylline 
but not NE is able to mobilize glucose and 
FFA. Preliminary studies indicate that theo- 
phylline increases the cyclic AMP content of 
liver about 6-fold. This suggests that the action 
of theophylline in mobilizing glucose is mediat- 
ed through cyclic AMP. The level of cyclic 
AMP in adipose tissue is also increased to some 
extent. 

Factors Which Affect Drug Action 

Embryotoxic Effects of Thalidomide 

The mechanism by which thalidomide exerts 
toxic effects on the embryo is still obscure. It is 
known that the drug is hydrolyzed nonenzymat- 
ically in the fetus to polar metabolites and it 
has been assumed, but not proved, that these 
metabolites are the cause of the embrytoxic 
effects of thalidomide. 

Our results show that labeled thalidomide, 
injected intravenously into rabbits (5 mg/kg), 
disappears with a half -life of about 2 hours. 



The lipid-soluble drug readily crosses the pla- 
centa at a rate that is presumably related to 
the plasma level. Accordingly, most of the drug 
that enters the fetus does so shortly after its 
injection and is hydrolyzed nonenzymatically 
to a variety of metabolites, which are highly 
polar and are trapped within the placental bar- 
rier. In 4 hours the concentration of total 
metabolites in the rabbit fetus reaches about 
100 fig/g, which is 500 times greater than that 
of unchanged thalidomide in fetal tissue, or 
plasma of mother (0.18 ;ug/ml). By giving the 
thalidomide intravenously, reproducible terato- 
genic effects are obtained in rabbits with doses 
ranging from 2.5 to 10 mg/kg (daily from the 
6th to the 10th day of pregnancy) . In contrast, 
data from the literature show that 4 hours 
after oral administration (100 mg/kg), the 
plasma level of the drug is about 4 /xg/ml and 
the total metabolites in fetus reach values equiv- 
alent to about 7 fig/g. Since Large doses given 
orally are necessary to cause teratogenic effects 
in rabbits, the embryotoxicity is apparently 
more closely related to the fetal level of metab- 
olites than to the level of the parent drug. 

These results indicate the importance of de- 
signing experiments with thalidomide that 
take advantage of the lipid solubility of the par- 
ent drug and the polar nature of possibly tox- 
ic metabolites. Since the metabolites in the 
fetus must originate from the parent drug, it is 
important that the parent drug attain a high 
plasma level in the mother. This is best done by 
intravenous injection. 

The literature shows that huge oral doses of 
thalidomide are needed in the rat to attain a 
teratogenic effect. Our results show that the 
drug has a potent teratogenic effect in rats 
after intravenous injection. 

Since the drug disappears in rats and rabbits 
at similar rates, these results suggest that the 
teratogenic effects in rats depend on the 
plasma levels shortly after i.v. injection. It is 
commonly believed that thalidomide and its 
metabolites cause malformations by interfer- 
ing with glutamic acid metabolism, but this 
may now be questioned since phthalimido- 
phthalimide, which does not contain a glutari- 
mide ring, is more embryotoxic than thalido- 
mide in rabbits. 



NATIONAL HEART INSTITUTE 



91 



Desmethylimipramine 

The cholinergic effects of tremorine are me- 
diated through the formation of oxotremorine 
and not by the parent drug. Last year we 
reported that desmethylimipramine (DMI) de- 
lays the onset of the tremors induced by tremor- 
ine, but prolongs the action of oxotremorine. 
We have now deiinitely established that DMI in 
rats affects the oxotremorine-induced syn- 
drome by inhibiting the metabolism of oxotrem- 
orine to inactive metabolites; pretreatment of 
rats with DMI impairs the metabolism of ox- 
otremorine by liver preparations. 

Enzymatic Mechanism of Drug Metabolism 

Evidence obtained in this and other laborato- 
ries indicates that the component in liver mi- 
crosomes, which reacts with Oo to form an 
"active oxygen" complex, involves a hemin 
pigment which can be identified by its ability to 
combine with CO to form a complex having an 
absorption maximum at 450 m/t. This pigment 
is called P-450. Carbon monoxide inhibits the 
oxidation of drugs by microsomes, as well as 
the oxidation of TPNH in the absence of drugs. 
In addition, ethylisocyanide which combines 
with P-450 is a good inhibitor. There is also a 
good correlation between binding of P-450 and 
inhibition of drug metabolism. Thus, the oxida- 
tion of drugs may be as follows. 

1. TPNH + oxidized P-450 

TPN-cytochrome c reductase 

-» 

TPN -I- reduced P-450 
l.a DPNH -1- oxidized P-450 

DPN-cytochrome c reductase 

— > 

DPN -1- reduced P-450 

2. Reduced P-450 + 0^ ±i? 0^ - P-450 

3. O2 — P-450 -I- Drug-^oxidized P-450 -t- 
oxidized drug 

Sum. TPNH (or DPNH) + 0^ + drug-^TPN 
(or DPN) + oxidized drug 

P-450 is reduced more rapidly by TPNH 
than by DPNH, which accounts for the appar- 
ent specificity of the enzymes for TPNH. In 
addition, P-450 is involved in the anerobic re- 
duction of nitro- and azo-compounds, since CO 



blocks the reduction of p-nitrobenzoic acid and 
prontosil. 

Compounds that block the drug-metabolizing: 
enzymes may interfere with P-450 in various 
ways. For example, incubation of mouse liver 
microsomes with a TPNH generating system 
and either SKF-525A or Lilly 18947 causes the 
formation of a substance, which has an absorp- 
tion maximum at 450 m/i, and is apparently an 
inactive form of P-450. JB 516 does not give 
rise to the 450 mju.-absorbing substances, but 
apparently destroys microsomal P-450 even 
without preincubation with microsomes. Fi- 
nally, DPEA, the primary amine analogue of 
Lilly 18947, apparently acts by inhibiting the 
reduction of P-450. 

Passage of Substances Across Membranes 

Central Nervoiis System 

An important problem concerns the mecha- 
nism by which the CNS disposes of polar metab- 
olites for the failure of such processes might 
lead to abnormal brain function. Our studies 
indicate that there is a saturable, active proc- 
ess in rabbits that rapidly transports choline 
and other quaternary amines. The site of this 
transport is the choroid plexus which in vitro 
accumulates the amine by a process having all 
the characteristics of active transport. 5HT, 
NE, hexamethonium, decamethonium, and 
NMN share this process, suggesting that the 
choroid plexus "pump" is important in dispos- 
ing of organic bases from the CNS. 

Further studies have been carried out with 
the active transport system in brain which 
transfers 5-hydroxy-indolacetic acid (5HIAA) 
directly from brain to blood. This process may 
be blocked by probenecid. The block is complete 
since the accumulation of 5HIAA in brain over 
a period of two hours is consistent with the 
known rate of formation. 

Preliminary results indicate that the ependy- 
mal membrane is highly permeable to choline, 
as well as hexamethonium, inulin, and manni- 
tol which pass from CSF to brain and presum- 
ably from brain to CSF by simple diffusion. 
This process is far slower than transport from 
CSF to blood. 



92 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Biliary Excretion of Drugs 

The liver is known to transport organic acids 
and organic bases into bile by diiferent sys- 
tems. A third transport process has been dis- 
covered that secretes ouabain, a nonionized 
compound, at a rapid rate (ti/a 13 minutes). Di- 
hydro-ouabain is also excreted like ouabain, but 
at a slower rate. This process is not inhibited 
by organic acids or bases. Preliminary results 
indicate that liver slices accumulate ouabain 
by an active transport process. 

Chlorothiazide, an organic acid lacking car- 
boxyl or sulfonyl groups, shares the same 
secretory process as other acids. Thus, the car- 
boxyl or sulfonyl acid groupings are not re- 
quired for transport. 

Enzymatic Mechanism of Mem,hrane Transport 
and Mechanism of Hormonal Control of 
Active Transport 

Since physiological control of the cardiovas- 
cular system involves operations on the cell 
membrane, the general theme will be to provide 
molecular descriptions of 1) the mechanism for 
maintaining excitability of nerve and muscle 
by the ion transport systems in the membrane 
and 2) the molecular changes in susceptible 
membranes caused by the impact of neurohu- 
moral transmitters and various drugs. 

Since there is abundant evidence that a 
membrane ATPase in nerve and muscle pro- 
vides the enzymatic machinery for ion trans- 
port, most current efforts are devoted to a 
study of the mechanism of this reaction within 
the limitations imposed by the impurity of par- 
ticulate preparations now available. An effort 
is being made to isolate proteins of sufficient 
purity for physical chemical studies of the 
structural changes which are presumably asso- 
ciated with the ion carrier function. With the 
exception of relaxation methods, which are not 
now being used in the NIH, the physical chemi- 
cal investigations of proteins will be carried 
out in collaboration with other groups. 

The identification of the phosphorylated in- 
termediate whose formation is catalyzed by so- 
dium, as a mixed anhydride of phosphoric acid 
and an acidic group of the protein, will permit 
rapid progress in studies of the active center of 
transport ATPase. The fact that this is a high 



energy intermediate suggests that the actual 
conversion of phosphate bond energy into os- 
motic work must be associated with the potas- 
sium-catalyzed dephosphorylation of the en- 
zyme. The high reactivity of the intermediate 
with amines and hydroxylamines should permit 
its ready conversion to a stable isotopically la- 
beled derivative that can be used to identify 
the particular amino acid that serves as the 
phosphate carrier. This work is now continu- 
ing. 

The second potassium-catalyzed part of the 
reaction may be studied independently of the 
sodium-sensitive intermediate formation by 
means of artificial substrates. This now makes 
it possible to determine by fractionation stud- 
ies whether the transport ATPase system is a 
single enzyme or a family of enzymes associat- 
ed on a matrix in the membrane. If the latter is 
the case, the use of artificial substrates may be 
the key to the isolation of pure components of 
the system. 

Stimulation of the synthesis of transport 
ATPase in the kidney of the adrenalectomized 
rat by corticosterone is still puzzling. Thus far 
the effect is unique for corticosterone, a hor- 
mone with very weak mineralocorticoid activ- 
ity in the rat. Efforts to relate the effects of 
the hormone to induced synthesis of protein 
are under way. It is anticipated that the isola- 
tion of pure protein components of the ATPase 
system will prove of value here, since it will be 
possible to demonstrate immunochemically 
whether the corticosterone stimulation is spe- 
cific for the protein of the ATPase system. 

Mechanism, of Uptake and Storage of Catechol- 
amines by Brain and Sympathetically In- 
nervated Tissues 

The uptake of catecholamines by an active 
transport system in the membrane of sympa- 
thetic nerves is well established as a mechanism 
which terminates the physiological effects of 
these amines. The newly taken up amines are 
almost instantaneously incorporated into stor- 
age granules. The biochemical processes in- 
volved in the transfer of catecholamine from 
the carrier site in the membrane to the storage 
granule are obscure. In the hope of clarifying 
this part of the uptake mechanism studies with 



NATIONAL HEART INSTITUTE 



93 



norepinephrine storage particles from heart 
were undertaken. 

Methods for the isolation of highly purified 
amine storage particles from rat heart have 
been devised and used in studies of the uptake 
of NE in vitro. Uptake by these preparations 
has been compared with uptake by cruder mi- 
crosomal fractions similar to those used in 
other laboratories. It appears that most of the 
in vitro uptake of NE previously ascribed to 
heart storage particles represents rapid ad- 
sorption onto a site that is neither specific for 
NE nor associated with the storage particle it- 
self. Much of the concentration of isotopic NE 
ascribed to these particles appears to represent 
the binding of metabolites. Isolated particles 
from heart, unlike those from splenic nerve or 
adrenal medulla, appear to have lost the ability 
to accept NE by processes related to the in vivo 
uptake. 

New Methods of Analysis 

An isotopic derivative technique for the as- 
say of secondary amines has been developed 
and applied to the assay of desmethylimipra- 
mine levels in plasma of patients receiving the 
drug in therapeutic dosage (25 mg per day). 
The results show that the plasma levels are as 
low as .030 microgm per ml and that there is a 
wide individual variability in the rate of bio- 
transformation. 

A procedure for the assay of labeled thalido- 
mide in plasma and tissues has been devised. In 
this method a simple procedure separates un- 
changed drug from its metabolic products. 

LABORATORY OF TECHNICAL DEVELOP- 
MENT 

Fluorescence Methods 

The development of methods for measure- 
ment of the polarization of fluorescence has 
shown that the Spectrophotofluorimeter (SPF) 
developed here can be modified to provide fluo- 
rescence polarization spectra of sufficient accu- 
racy to permit application of the corrected 
spectra to the characterization of protein-dye 
interaction and intramolecular fluorescence in- 
teraction. 



It has been shown that when the SPF is 
equipped with a recently developed ultraviolet 
polarizing filter (Polacoat) set, polarization 
errors, introduced by the use of grating mono- 
chromators, can be eliminated from the meas- 
urements. In addition, a relatively simple cor- 
recting procedure developed elsewhere has 
been used to provide the spectral energy versus 
wavelength correction. The effectiveness of the 
method is shown by the agreement of our data 
with values obtained by others in quantum 
yield measurements and in close agreement of 
our data with theoretical values of polarization 
ratios obtainable from dyes in high viscosity 
solvents. The ability of the technique to indi- 
cate the characteristic conformation of the pro- 
teins and the interaction of bound dyes was 
demonstrated by the correlation of the known 
allosteric behavior of glutamic dehydrogenase 
(GDH) with results obtained with the fluores- 
cence depolarization method. 

Suitable dyes were prepared, purified and 
methods of dyeing and separating the product 
were developed and measurements made. Dye 
enzyme complexes were still highly active and 
the measurements indicated free rotation of 
some segments of the molecule. The molecule 
does not behave as a compact ellipsoid as would 
be required for determination of axial ratios by 
fluorescence depolarization previously reported 
by Weber. 

The system of GDH and DPNH studied by 
the native fluorescence of the tyrosine and 
tryptophan indicated no heterogeneity of the 
four DPNH binding sites. The fluorescence po- 
larization of the tryptophan and tyrosine were 
altered by urea and detergents in agreement 
with expected behavior. In the observation of 
the GDH activity, photo-sensitivity of the en- 
zyme required the development of method to 
reduce the photo-degradation. Published re- 
sults of these measurements describe the tech- 
nique of reducing the effect enough to permit 
accurate measurements. It was also shown that 
this inactivation by monochromatic light was a 
phenomenon distinct from the inactivation by 
x-rays studied elsewhere. 

Studies of GDH using quantum yield and 
fluorescence polarization with dyes, urea, de- 
tergents and allosteric regulators are continu- 
ing. The binding of acridine dyes to DNA has 



94 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



been studied elsewhere because these dyes are 
known to be mutagenic and because it was pro- 
posed that the mode of binding is related to the 
mutagenic activity. The mutagenic effect was 
explained by Lerman and Luzatti some years 
ago as owing to coding errors resulting from 
intercalated binding of the dye. As circular 
dichroism and spectral titration data were not 
consistent with the theory, fluorescence polari- 
zation studies were undertaken in cooperation 
with the NIMH physical chemistry laboratory. 
DNA Acridine Orange (AO) complexes were 
examined over a range of solution viscosities 
by utilizing the modified SPF. The bound AO 
molecules were shown to have degrees of free- 
dom inconsistent with the intercalation theory. 
Som additional dye-dye interaction which re- 
sults in a red fluorescence which can be studied 
with the new red sensitive detectors recently 
made available for the SPF was also observed. 

A microspectrofluorometric system for the 
study of intracellular tetracycline in micro-or- 
ganisms has been developed for the Laboratory 
of Biology of Viruses of NIAID. The system 
permits satisfactory measurement of the inten- 
sity of fluorescence from selected one micron 
regions of observation. An EMI type 9524S 
multiplier phototube provides the extreme sen- 
sitivity required with a dark current of only 3x 
10"^^ A or a 50 to 100 fold signal/noise advan- 
tage over the usual 1P21 tube. A fluorescence 
micro-method for inulin is under development 
with sensitivity of 5 nanograms. The method 
offers advantages of simplicity and sensitivity 
over the currently used colorimetric method. 
The reaction of 5,5: dimethylcyclohexane 1, 3 
dione (dimedon) with fructose in 85% 0-phos- 
phoric acid to form a fluorescent product is the 
basis for the method. Activation at 360 m;a and 
fluorescence at 410 m;ii was established with 
the SPF and a special purpose filter instrument 
suitable for reading microcuvettes was con- 
structed. Limitations currently seem to be relat- 
ed to non-uniform fluorescence of the dispos- 
able microcuvettes. 

Ultramicro Methods 

The helium glow photometer has now been 
developed into a practical instrument that per- 
mits the simultaneous analysis of Na and K 



content (Picomoles) in submicrogram amounts 
of kidney tubule tissue to ± 5%. 

During the development it was necessary to 
determine the mutual interaction of the alkali 
metals as well as anion effects and develop a 
suitable diluent to reduce the errors due to 
these interactions. A diluent containing cesium 
and phosphate has been satisfactory to 
"swamp out" potential errors due to enhance- 
ment or inhibition of the characteristic emis- 
sion. The use of a dichroic mirror separates 
the Na and K emission and high efficiency mul- 
tilayer interference filters select the lines for 
measurement by two multiplier phototubes. 
The photocurrent of each tube is integrated 
electronically for a specific time to quantify the 
content of Na and K. 

Limitations of the method have been shown 
to be related to sample handling by comparison 
with radioactive sodium delivery with the same 
methods. The use of newly developed pipettes 
for nanoliter transfer and special coatings of 
Kel-F have facilitated the necessary transfers. 

A micro-pipette for automatic filling and 
transfer of nanoliter quantities was developed 
and a description published. The measuring pi- 
pette was constructed of quartz and sealed by 
flame fusion into a larger glass capillary. The 
use of quartz permits the use of a micro-flame 
to produce the seal without distorting the 20 to 
30 /x ID measuring capillary and the extremely 
small size permits direct quartz to glass fusion 
without cracking. Modifications with bulbs for 
several nanoliters have been constructed; this 
modification permits the simultaneous observa- 
tion of both ends of the pipette in the 60x mi- 
croscope field. 

An ultramicro freezing point depression ap- 
paratus and method previously described was 
completed and the results published. Efforts 
are being continued to optimize the equipment 
by utilizing commercially developed packages 
for coolers and for electronic control apparatus 
in order to derive a practical apparatus that 
will be useful to other laboratories. One instru- 
ment manufacturer has undertaken to produce 
a commercial instrument and another has indi- 
cated interest in doing so. The laboratory pro- 
totype has been used to establish the feasibility 
of the method. A commercial model for evalua- 
tion is expected in the near future. 



NATIONAL HEART INSTITUTE 



95 



Micro apparatus required for the perfusion 
and handling of isolated functional sections of 
renal tubules stimulated the development of 
special controlled thermoelectric cool stages 
and controlled temperature chambers. A perfu- 
sion pump based on the controlled expansion of 
a liquid by a programmed, servo-controlled 
heating schedule delivers from 5 to 50 nano- 
liters (10-^) per minute from a pumphead 
containing no moving parts. The head is 1 cm. 
in diameter by 3 cm. long and is suitable for 
mounting directly on the manipulator and hold- 
ing the perfusion pipette with a minimum of 
total volume or dead space. The design pro- 
vides freedom from expansion effects due to 
ambient temperature changes. 

Artificial Organs 

Blood Oxygenator 

Production of our disposable membrane ar- 
tificial lung undertaken by the Dow Corning 
Company is proceeding at a slow pace with 
their development of suitable semi-automatic 
machinery for mass production of these units. 
The mass production is reported to be undergo- 
ing preliminary test at the moment. It is of in- 
terest to note that the General Electric Com- 
pany has now succeeded in making a pin hole 
free silicone membrane only 1 mil thick and the 
final design of our membrane lung is entirely 
dependent upon the membrane thickness that is 
to be used. A thin membrane will permit a 
smaller area to be used with consequent reduc- 
tion in size and priming volume. It is presumed 
that the advantages of this new development 
will be included in the production of a dis- 
posable lung. A few preliminary production 
samples have been received but as these do not 
represent the final production device they were 
not considered useful for extended animal test- 
ing. Continued development of the pump to be 
incorporated inside of the artificial lung is be- 
ing continued with development of suitable 
valves and pumping ventricles. Information ob- 
tained from the biventricular assistor will be 
utilized in this development. 



Blood Pumping 

A method of cardiac assistance which pro- 
vides a squeezing action to both ventricles of 
the heart is now in the testing stage. In this 
method a close fitting rubber envelope is placed 
around both ventricles so that at rest the 
rubber envelope maintains the heart in a sys- 
tolic condition. This envelope, suitably con- 
trained by a rigid lexan container, is expanded 
to the diastolic condition by the introduction of 
suction between the rubber envelope and the 
outer lexan shell. The rubber envelope is thus 
stretched by the unbalance of pressures created 
by the suction. This rubber envelope is heavy 
enough so that upon release of suction it snaps 
back to its normal relaxed resting condition 
producing the ejection force necessary to pump 
the blood. Design features of this assistor per- 
mit rapid application of the device by applicat- 
tion of reduced pressure to the interior of the 
squeezing or active rubber envelope to suck the 
heart into place, and, when necessary, the 
heart can be popped out by similar applications 
of slightly increased pressure. The use of the 
close fitting ventricle and the constricting ac- 
tion of the rubber envelope around the ven- 
tricle maintains the assistor in position with- 
out the necessity for any clamping or tethering 
device. A lining of silicone jelly helps to main- 
tain the assistor in position and protects the 
surface of the heart from abrasion. Animal 
tests have shown that this device remains in 
place and is capable of supporting the circula- 
tion with the ventricles fibrillating for periods 
up to an hour. When the fibrillating heart was 
defibrillated normal heart function returned. 
Testing of the cardiac assistor will be contin- 
ued with the expectations that it may be of use 
in supporting the circulation under various 
clinical emergency situations such as acute 
heart failure produced by coronary occlusion or 
for support of the circulation following cardiac 
surgery when a normal beat cannot be re- 
stored. Under these circumstances temporary 
support of the circulation could be maintained 
until the heart recovered. It is also conceivable 
that some such arrangement might be used as a 



96 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



more permanent installation to support the cir- 
culation either indefinitely or until a suitable 
prosthetic device could be applied, the tempo- 
rary support providing the delay necessary for 
the scheduling and production of custommade 
equipment for the particular individual. 

Blood Dialyzer 

Utilizing principles of construction and effi- 
cient perfusion of a membrane developed with 
the work on the artificial lung, an effective ar- 
tificial kidney was produced and tested. A low 
priming volume and low perfusion pressure 
with an effective method of stirring the mem- 
brane surface film, resulted in a device with 
very effective near theoretical performance 
characteristics. As this device incorporated sim- 
ilar production requirements and marketing 
facilities as the membrane lung, the Dow Corn- 
ing Corporation is considering the marketing 
of this device. It is anticipated that this may be 
suited for home dialysis because of its simplic- 
ity and economy. Further experimental work 
will be continued when a commercial model of 
this dialyzer becomes available to us for test- 
ing. 

Fast Reaction Methods 

The development of instruments and meth- 
ods for the study of fast reactions include im- 
provements in time resolution, and reduction 
of interfering cavitation effects. Several sys- 
tems which have been assembled and tested 
permit simultaneous observation of optical 
density changes and heat production with a 
time resolution of 2 to 3 milliseconds and ther- 
mal resolution of 2 x 10"= calories. In this 
system temperature equilibrium is accom- 
plished in IV^ hours, minimal quantities of 
reactants are required (2 ml per observation) 
and 99 percent mixing occurs in about 0.4 mil- 
liseconds. 

A complete instrument constructed by 
Science Products, incorporating several new 
design concepts, has been completed for us and 
provides finer time resolution by driving the 
pistons of concentric syringes with a pulse of 
high pressure gas. The rapid acceleration 
through the mixer into the observation 



chamber improves the time resolution to 0.2 
milliseconds and a new stopping valve for 
stopped-flow operation stops the flow in 0.1 
millisecond. Eeactant requirements have been 
reduced to 0.5 ml. Fast flow monitoring has 
been facilitated by the development of a DC 
magnetic flowmeter with reduced polarization 
artifact. 

High speed mixers required for the efficient 
mixing of the reactants should minimize ther- 
mal and pressure changes at the jets. Reduc- 
tion of these effects with high speed mixing 
devices has been proceeding with the con- 
struction of a variety of geometrical arrange- 
ments of jets and observation tubes which are 
tested utilizing the gas-operated syringe 
drivers. Observation of the mixing tube with 
high speed photography is used to determine 
the points of cavitation, streaming and turbu- 
lence to optimize the design. 

Several fast thermal detectors, such as the 
copper-doped germanium and the strontium- 
doped barium titanates, have been obtained 
and are being evaluated with regard to sensitiv- 
ity and speed of response. 

A combination absorption or fluorescence 
stopped-flow apparatus which permits a reac- 
tion time curve to be determined on 0.2 ml of 
each reagent has been constructed. Time reso- 
lution is 2 milliseconds. Eeaction mixtures of 5 
millimolar aldolase and dihydroxyacetone phos- 
phate have been measured at 245 m/x and 10"^ 
molar bovine serum albumin. 

Work is in progress on the determination of 
heats and rates of reaction of cobaltioxalate 
ion and ferrous ion for use as a rapid flow ap- 
paratus calibration reaction. In order to clarify 
some points in the theory of hemoglobin oxy- 
genation reactions, thermal measurements of 
the first oxygen reaction with hemoglobin are 
being made with the continuous flow thermal 
apparatus since 0.4 milliseconds time resolu- 
tion is needed. 

A twin cell differential micro-calorimeter 
has been constructed for the study of the heats 
of reaction of biochemical and cellular systems. 
A mixer with a special low heat of mixing has 
been developed so that 2 to 3 ml of one solution 
may be mixed with 0.1 to 1 ml of another solu- 
tion and a total heat of 10 millicalories meas- 



NATIONAL HEART INSTITUTE 



97 



ured to 2%. No thermostat is needed for work 
at any room temperature which is constant to 
plus or minus 1 degree C. By using external 
thermostating a temperature range from — 20 
to 100 degrees C has been obtained in a now 
available commercial version. For static heat 
capacity measurements using an electrical 
heater, stabilities of 5 microcalories have been 
achieved. Work is being done at present on the 
heat of reaction of ATPase in red blood cells. 

Gas Chromatography 

The development of instrumentation for gas 
chromatography is being continued along sev- 
eral lines utilizing the dependence of velocity 
of ultrasound on gas composition. The original 
system which involved vacuum tube circuits 
has been redesigned and constructed using sol- 
id state circuits. The detector volume of a few 
microliters, high temperature tolerance, non- 
destructive detection, elimination of radioactiv- 
ity and predictable physical behavior over a 
definable range has made the system of suffi- 
cient interest that the Micro Tek Corporation 
has based an instrument on our published re- 
sults and has independently improved the 
detector cell to permit a greater temperature 
range and higher stability. It is expected to ap- 
pear on the market soon. 

This detection system is also directly appli- 
cable to analysis of binary gas mixtures with- 
out gas chromatography. As the cell dimen- 
sions and frequency determine the sensitivity 
limits, calibration can be made in physical 
terms without the need for repeated calibra- 
tions. It is expected that absolute and perma- 
nent calibrations can be made to 0.10%. 
Apparatus to provide gas mixtures of sufficient 
accuracy to demonstrate the capabilities of this 
system over the range of applicability has ne- 
cessitated the development of a gas mixing 
pump which will be completed in the next four 
weeks. Low gas flow measurements for mixing 
and calibration of similar systems has generally 
utilized the soap bubble flowmeter but permea- 
bility of the film, chemical interaction, or solu- 
bility of the gas limits the applicability or this 
meter to several gases of interest, i.e., NH4 and 
CO2. A dry sealed piston that follows the flow 
in response to minute changes in pressure has 



been tested as a slow flowmeter and a complete 
instrument which provides flow measurements 
for flows less than 100 cc/minute with a repeat- 
ability of 0.03 % has been developed. 

Applications of the ultrasound velocity gas 
chromatography detector have included the 
analysis of respiratory gases in cooperation with 
the cardiology branch. One to two ml samples 
of air are analyzed every 20 to 30 seconds utili- 
zing a 2" silica gel column with nitrogen carrier 
gas. The method provides the data for rapid 
repetitive RQ measurements. Another system 
is being applied to H2 and CO™ production 
for bacterial metabolism studies in a coopera- 
tive study with the Laboratory of Microbiology 
of the National Institute of Dental Research. 

New developments in capillary column liquid 
chromatography as reported by Beyer are lim- 
ited in sensitivity by the effluent detector 
system. Ultrasound velocity methods can be 
applied to liquid systems by utilizing higher 
ultrasound frequencies and capillary paths. Pre- 
liminary tests have indicated a suitable fre- 
quency, cell size, and a sensitivity more than 
adequate for applications to the detection of 
amino acids separated on liquid capillary col- 
umns. Nanogram sensitivities and in 2 to 20 ;u,l 
volumes are predictable on the basis of obser- 
vations in larger cells. 

As the dielectric constant of the gas from the 
gas chromatograph changes with eluted frac- 
tions this change has been detected by the use 
of paired capacitors that alternately determine 
the frequency of a high frequency oscillator. 
Continuous measurement of the frequency with 
a discriminator produces a signal related to 
changing dielectric constant. The simplicity of 
the cell and circuitry are the main features. 
Refinements of the circuit to eliminate the 
switching noise will be necessary before eval- 
uation can be completed. 

Blood Flowmetering 

An ultrasonic flowmeter which measures ul- 
trasound velocity upstream and downstream si- 
multaneously has been shown to have superior 
theoretical capabilities over systems proposed 
and published elsewhere. There remains some 
baseline drift to be corrected by the develop- 



98 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ment of a suitable phase lock system. Tests on 
2 mm diameter crystals separated by 15 mm 
showed linear, reproducible, bi-directional re- 
sponse. Adaptation of these elements to a satis- 
factory catheter tip probe remains to be accom- 
plished. A variant of the ultrasound system 
requiring only a single crystal has had limited 
success due to the low level of reflected signal 
from the blood cells and unknown effects due to 
turbulence in the region of the transducer. 

Nuclear magnetic resonance flow methods 
have been shown to have adequate sensitivity 
but it is not yet clear how to employ the capa- 
bilities of the physical interaction of the mag- 
netic hydrogen nucleus to construct a practical 
flow metering system for biological observa- 
tion. Electronic equipment for testing several 
of the possibilities is nearly complete and will 
be used with a new magnet to determine the 
limitations and advantages of the various mod- 
els. 

Mathematics and Computers 

1. A computer method of analysis which 
permits the physical laws governing conduc- 
tion or diffusion to be expressed in first-order, 
finite form has been developed. Solutions are 
obtained in one and two dimensions for rec- 
tangular, cylindrical, and spherical geometries 
without the use of transcendental functions or 
other conventional mathematical iterative 
methods. The case of the cylindrical calori- 
meter with concentric insulating and conduct- 
ing layers of varying chemical properties and 
with distributive heat sources arising from 
biochemical reactions at the center is treated in 
the transient and steady-state case. Work is 
being extended to three dimensions and the 
problem of cell membrane diffusion as well 
as consecutive and simultaneous chemical 
reactions. The problem of data handling as it 
comes from the detector is being solved by digi- 
talizing using a Computer of Average Tran- 
sients (CAT) or a hybrid control data 3100 
computer. Data from the mass spectrometer 
and stopped-flow reaction apparatus are being 
programmed. 

Retention index systems have been utilized 
for qualitative component identification in gas 



chromatography. Incompletely resolved com- 
ponents exhibit apparent displacement of 
retention index. According to theory, this dis- 
placement results from (1) the simple summa- 
tion of two unresolved distribution functions 
and (2) solute interaction. A repetitive analog 
computer developed in the Laboratory of Tech- 
nical Development was utilized to investigate 
the contribution of solute interaction for 
sample sizes utilized in analytical scale gas 
chromatography. The results of our investiga- 
tions demonstrate that for samples smaller 
than 100 micrograms (as normally employed 
for analytical scale separations) no significant 
variation in peak position occurred due to sol- 
ute interaction. It was further found that the 
repetitive analog computer is an effective aid 
in the quantification of partially resolved gas 
chromatographic peaks (3). 

It has been demonstrated mathematically 
that counterflow systems with active transport 
out of the ascending limbs in only the outer 
renal medulla cannot give the known sodium 
concentration profile in the inner medulla. The 
importance of these results is that it is possible 
to rule out a large class of systems as possible 
concentrating devices. 

LABORATORY OF CARDIOVASCULAR 
PHYSIOLOGY 

Net Potassium Changes in the Heart Associated 
with Altered Contractility 

It had previously become clear that if the 
effects of pharmacologic interventions on the 
K+ balance of the heart are to be fruitfully ex- 
amined, the hemodynamics of the heart, espe- 
cially aortic pressure and heart rate, must be 
maintained constant since alterations in these 
parameters will affect net K+ balance. The iso- 
lated supported heart preparation therefore 
continued to be the experimental preparation 
of choice. The overall objective of these studies 
was to gain further insight into the working 
hypothesis that quantitatively small shifts in 
K+ are importantly related to the contractility 
changes seen during homeometric autoregula- 
tion and certain pharmacologic interventions. 



NATIONAL HEART INSTITUTE 



99 



The following table of consolidated data sum- 
marized the findings to date. 



Myocardial 
Intervention Potassium 

Aortic Pressure 

Increased — 
Heart Rate 

Increased — 
Paired Stimulation — 


MnocardiaZ 
Contractility 

+ 

+ 


Ox^/gen 
Consumption 

+ 

+ 

+ 


Acetyl 

Strophanthidin 
Quinidine Sulfate 
Pentothal Sodium 
Potassium Chloride 


+ 
+ 
+ 


+ 









Norepinephrine 
Calcium Chloride 


+ 
+ 


+ 
+ 







+ = net K+ gain 

— = net K+ loss 

+ == increased contractility 

— = decreased contractility 

+ = increased O2 consumption 
= no change in O2 consumption 

From these data a consistent pattern of rela- 
tionships has emerged. 

A. Those interventions below the solid line 
(norepinephrine and calcium) do not influence 
contractility by way of a net K+ loss since the 
increase in contractility they induce is accom- 
panied by a net gain of K+. 

B. With the four interventions shown be- 
tween the solid and dashed lines, a net gain 
K+ is associated with a decreased ventricular 
contractility and a net loss of K+ with an in- 
creased contractility. With acetyl strophanthi- 
din, analysis of the fourteen experiments in 
which this agent was administered showed a 
good correlation between the dose given and 
the net K+ lost. It will be recalled that small 
doses of acetyl strophanthidin produce a small 
contractility increase and large doses produce a 
large contractility increase. There is, therefore, 
an important correlation between net K+ loss 
and contractility increase with this agent. 

C. Three hemodynamic interventions (in- 
cluding paired stimulation) were made. These 
are shown above the dashed line in the table. In 
each instance, a period of increasing contractil- 
ity occurred as a consequence of the interven- 
tion. In each instance a net loss of K* occurred. 
The range of these net K* losses was compa- 
rable to the range of net K+ losses observed 



with inotropic doses of acetyl strophanthidin. 

Studies were continued quantifying the up- 
take of K+ by the yeart when KCl is infused 
in amounts which elevate left ventricular end- 
diastolic pressure. The amounts of K+ uptake 
under such circumstances were comparable to 
those seen to be lost from the heart during the 
interventions above the solid line in the table. 
On the basis of the above results, a reason- 
able conclusion seems to be that contractility 
and K+ changes are importantly related. The 
possibility existed, however, that when a rise 
in left ventricular end-diastolic pressure oc- 
curred during infusion of K+, that this was 
produced by altering conduction and therefore 
synchronicity rather than the contractile fiber 
event itself. Data have become available rela- 
tive to the last point. As noted above, with the 
onset of paired stimulation a marked increase 
in contractility occurs. A net loss of K+ in 
amounts at least as large as those seen after a 
large dose of acetyl strophanthidin is also seen. 
When paired stimulation is terminated, con- 
tractility decreases while a net gain of K+ is 
taking place. Observations on conduction be- 
fore, during and after paired stimulation do 
not reveal conduction to be altered. These data 
indicate that if the net K+ changes are in- 
fluencing contractility, they are not doing so by 
altering conduction.. 

Further data have been gathered on net K+ 
losses during homeometric autoregulation in- 
duced by abrupt increases of aortic pressure 
while stroke volume and heart rate are held 
constant. This was done with the objective of 
having more complete information about the 
relation between the net K+ losses observed 
and the simultaneous increases in O2 consump- 
tion. It has become clear from the analysis of 
twenty-two such experiments that the greater 
the increase in oxygen consumption the greater 
will be the net loss of K+. Some means of ascer- 
taining whether these are simple concomitants 
or causally related phenomena will be the sub- 
ject of continuing investigation. With refer- 
ence to this point an interesting dichotomy is 
apparent in the table. The pharmacologic inter- 
ventions investigated uniformly produced net 
K+ changes without oxygen consumption 
changes. No hemodynamic intervention was ob- 
served to produce a net K+ change unless a 
change in oxygen consumption did occur. 



IQO 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



It had been reported by others who, in our 
view, used less rigorously controlled and pre- 
cise techniques, that insulin and nethalide 
would dissociate the net loss of K* from the 
increase in contractility when acetyl stro- 
phanthidin is given. It was, therefore, felt desir- 
able to repeat such experiments. Our data do 
not confirm the observation that the prelimi- 
nary administration of either insulin or nethal- 
ide diminishes either the net K+ loss from the 
heart or the increase in contractility when ace- 
tyl strophanthidin is administered. 

Myocardial Oxygen Consumption 

Previous work from this laboratory intro- 
duced the advantages of relating the oxygen 
utilization of the heart to the amount of ten- 
sion developed by ventricular myocardium at 
least insofar as this is reflected by the area 
under the systolic portion of the ventricular 
pressure curve (tension time index) . This rela- 
tionship was seen to hold for any given state of 
the myocardium. 

Two additional bodies of data relative to the 
general problem of myocardial oxygen con- 
sumption in the pumping heart have become 
available. The first is that with pharmacologi- 
cally induced increases in contractility in addi- 
tion to the fall of left ventricular end-diastolic 
pressure, there is a decrease in the tension time 
index with no change in oxygen consumption. 
With agents that decrease contractility, the 
tension time index is increased with no change 
in oxygen consumption. In the new contractile 
state, the oxygen consumption will once again 
vary with hemodynamically induced changes in 
the tension time index. There is, in short, a 
family of curves describing this relationship 
and any particular curve can be shifted by 
inotropic influences much as inotropic influ- 
ences shift the curve relating end-diastolic 
pressure to stroke work. 

The second type of generality that became 
apparent was that if aortic pressure, stroke vol- 
ume and heart rate are held constant, inotrop- 
ic agents can markedly influence the maxi- 
mum rate of rise of ventricular pressure with 
no change in oxygen consumption. Only with 
one of these (norepinephrine) was a significant 
increase in oxygen consumption observed and 



this occurred only when very high 'doses of the 
drug were given. Over a large part of its dos- 
age range, left ventricular end-diastolic pres- 
sure fell, the duration of ejection shortened, 
and the maximum rate of rise of ventricular 
pressure increased with no change in oxygen 
consumption. These data indicate that when 
oxygen consumption is increased as the result 
of inotropic interventions, such an increase 
cannot be attributed solely to the increase in 
the maximum rate of rise of ventricular pres- 
sure. 

It was the complicated geometry of the in- 
tact pumping heart that made necessary the 
use of a tension index instead of a direct calcu- 
lation of tension. Two approaches have been 
utilized to effect a transition from this index to 
a measured value. The first is in a preliminary 
stage and consists of making simultaneous de- 
terminations of pressures, absolute dimension- 
al changes and oxygen consumption. The sec- 
ond approach has yielded a satisfactory body 
of data from experiments in which the oxygen 
consumption of papillary muscle, with its rela- 
tively uncomplicated geometry, has been ex- 
amined. An inverse relationship between veloc- 
ity, shortening and oxygen consumption was 
seen. The expected lack of correlation between 
oxygen consumption and external work is also 
seen in papillary muscles. Isometric studies 
demonstrated the lack of a direct relationship 
between changes in initial tension, total tension 
area, and contractile element work and oxygen 
consumption. Only the developed tension and 
developed tension area were found to correlate 
with changes in oxygen consumption. 

Factors Influencing Peripheral Vascular Re- 
sistance During Exercise 

The possible influence of potassium on the 
vascular resistance during muscular activity 
was extensively investigated in the isolated, 
perfused, canine gracilis muscle. The first 
phase of this study consisted of studying the 
net K+ loss from the muscle associated with the 
transition from rest to exercise. 

The data show that venous K+ concentra- 
tions increase during muscular contraction and 
that the greater the number of contractions the 
greater the increase in venous K*. The same 



NATIONAL HEART INSTITUTE 



101 



relationship holds with arterial inflow occlu- 
sion; i.e., the longer the duration of occlusion, 
the greater is the venous K+ level upon restora- 
tion of blood flow. Signiflcantly, the degree and 
particularly the duration of decreased vascular 
resistance correlated with the degree of muscu- 
lar activity and the duration of arterial occlu- 
sion. Thus, there was also a correlation be- 
tween the resistance change and the increase in 
venous K*. While this correlation was main- 
tained in all experiments during the period 
immediately following contractions and follow- 
ing return of blood flow, it was not infre- 
quently observed that resistance could return 
to control levels and venous K+ remain elevated 
or vice versa. Further, the extent of the K+ ele- 
vation was quite small (0.2 to 0.8 mEq/L) . In- 
fusions of even much larger amounts of K+ 
failed to produce a decrease in vascular resist- 
ance that approached that decrease observed 
with the muscular contraction or arterial occlu- 
sion. 

Because it was not possible to effect the same 
degree of vasodilation in the muscle vascular 
bed by infusing an amount of potassium that 
produced the same or even higher venous K+ 
level as that associated with exercise and with 
the arterial occlusion data in view, experiments 
were designed to determine if a given level of 
K+ might not have a varying vasodilator effect 
at different O2 levels. 

These experiments yielded data showing that 
perfusion with high O2 blood containing low lev- 
els of K+ (1.0-2.0 mEq/L) will result in an 
increase in vascular resistance. Perfusion with 
low O2 blood (1.0-8 vol.%) containing low lev- 
els of K+ (1.0-2.0 mEq/L) does not produce 
an increase in resistance but rather a de- 
creased vascular resistance. Increasing the K* 
concentration of the perfusing blood, whether 
of high or low O2 content, results in a progres- 
sive decrease in vascular resistance, the reduc- 
tion being greater with each increasing K+ level 
with the low O2 blood than with blood of high 
O2 content. Resistances approaching those pro- 
duced by exercise have not been realized even 
at extremely high levels of K+ when the muscle 
is perfused with high O2 blood. It has, however, 
not been unusual to reach "maximum vasodila- 
tion" when the muscle is being perfused with 
low O2 blood containing K+ in concentrations 



well within the venous K+ ranges observed as a 
result of inducing muscular contractions. The 
data obtained thus far indicate that blood O2 
and potassium levels are importantly related to 
the degree of tone maintained by the resistance 
vessels in skeletal muscle and that these factors 
interrelate and reenforce each other in their in- 
fluence on peripheral vascular resistance in the 
transition from rest to exercise. 

The powerful vasodilator action of the kalli- 
dins has in the past given rise to the suggestion 
that these substances may contribute to the de- 
crease in vascular resistance associated with 
muscular activity. Investigation of this possibil- 
ity was made in the isolated, perfused (con- 
stant-flow) gracilis muscle of the dog. In those 
experiments where carboxypeptidase B was 
measured in lymph, the lymph was collected 
from the main lymphatics during gentle exer- 
cise of the hind limb of the dog. Infusion of 
kallidin-9 at 1-4 /ig/min directly into the gra- 
cilis artery consistently produced a pronounced 
decrease in resistance which could be effec- 
tively blocked by simultaneous perfusion of the 
muscle with blood containing 15-60 /tg/ml hog 
pancreatic carboxypeptidase B. The vasodilata- 
tion produced by muscle contraction, arterial 
occlusion and lumbar cholinergic sympathetic 
nerve stimulation was not altered by the pres- 
ence of carboxypeptidase B. The failure of 
carboxypeptidase B to block the three interven- 
tions could have been due to its absence from 
perivascular fluid. However, this enzyme, with 
a molecular weight of 34,300 was found in hind 
limb lymph following systemic infusion in a ra- 
tio similar to that of the plasma proteins. Also, 
the vasodilatation following occlusion was 
more prolonged when the occlusion was imme- 
diately preceded by muscle contraction than 
when preceded by kallidin infusion, indicating 
that the polypeptide was not as stable as the 
vasoactive metabolites or metabolite released 
during exercise. 

The failure of carboxypeptidase B to alter 
the decrease in vascular resistance following 
muscle contraction, arterial occlusion and lum- 
bar cholinergic sympathetic nerve stimulation, 
the presence of carboxypeptidase B in perivas- 
cular fluid, and the greater stability of the ex- 
ercise factor as compared to kallidin make it 
unlikely that activation of the kallikrein-kalli- 



102 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



din system is responsible for these types of va- 
odilation in contracting skeletal muscle. 

Renal Studies 

Intravascular Receptors in Water and 
Salt Regulation. Previous studies from 
this laboratory have shown that the diuresis 
associated with acute isotonic iso-oncotic in- 
travascular volume expansion is attenuated by 
either acute bilateral carotid sinus denervation 
or acute bilateral cervical vagotomy; sodium 
excretion is however not influenced. As a con- 
uation of these studies experiments have been 
undertaken to determine the influence of 
chronic cardiac denervation upon the diuretic 
and natriuretic responses to intravascular vol- 
ume expansion. The renal responses of a group 
of dogs to volume expansion were determined 
prior and subsequent to chronic cardiac dener- 
vation. The results of the experimenta have 
shown that the absence of cardiac nerves sig- 
nificantly diminishes both the diuretic and na- 
triuretic responses to acute volume expansion 
apparently independently of changes in glo- 
merular filtration rate. These experiments, tak- 
en in conjunction with the earlier acute vagot- 
omy experiments have led us to the position that 
whereas cardiac vagal afferent nerves appear 
to modulate free water excretion, cardiac sym- 
pathetic nerves may modulate sodium excre- 
tion. The cardiac sympathetic nerves concerned 
appear to be afferent fibers since (1) any in- 
fluence of sympathetic efferent nerves would be 
expected to be reflected by changes in filtered 
sodium secondary to changes in G.F.R. (2) The 
drug tests used to verify denervation indicate 
that one of the animals had regained efferent 
but not afferent innervation (failure to re- 
spond to veratramine with bradycardia) . This 
animal continues to show an attenuated diu- 
retic and natriuretic response to acute volume 
expansion. The chronic denervated dogs usual- 
ly have completely regained innervation twelve 
months after the operation. The animals in the 
present series will be followed to determine if, 
after this time, the normal natriuretic and diu- 
retic responses to acute volume expansion 
returns. 

It has been reported that section of the vagi 
in the normal dog undergoing water diuresis is 



associated with antidiuresis. It has been as- 
sumed that this response was secondary to the 
section of atrial afferent nerves. In order to de- 
termine the influence of extracardiac vagal 
afferents upon water diuresis, studies were 
done employing the chronic cardiac denervated 
dog. Water diuresis was established in this 
preparation and the influence of bilateral cer- 
vical vagotomy determined upon free water 
clearance, osmolal clearance, inulin clearance 
and sodium excretion. Bilateral cervical vagot- 
omy was always associated with a reduction in 
urine flow which was not consistently related 
to changes in osmolal and inulin clearance or 
sodium excretion. Free water clearance always 
declined, the maximum decrease occurring ap- 
proximately 15 minutes after vagotomy. In 
some experiments the antidiuresis was well 
maintained. In other experiments an escape 
phenomenon was seen in that after the initial 
reduction, urine flow again increased to or to- 
wards the control level. In one experiment su- 
pradiaphramatic vagotomy was done. Section 
of the vagi at this level had no influence on any 
of the variables measured while cervical vagot- 
omy produced the characteristic antidiuresis. 
These experiments support the position that 
receptors which modulate free water excretion 
are contained in the pulmonary bed. It is pos- 
sible that the receptors concerned are osmore- 
ceptors. 

To determine further the influence of car- 
diac nerves on sodium excretion, experiments 
have been initiated to determine the natriuretic 
and diuretic response of the chronic cardiac 
denervated dog to acute saline loading. Al- 
though preliminary, the experiments suggest a 
pattern of response which is different from the 
dog which has its cardiac nerves intact. 

The Relation of the Kallikrein System 
TO Renal Function. Earlier studies from 
this laboratory demonstrated that kallidin is a 
potent renal vasodilator and produces an in- 
crease in glomerular filtration rate, water and 
electrolyte excretion. It was further observed 
that these changes could be mimicked by ace- 
tylcholine, and that during the intrarenal infu- 
sion of acetylcholine the level of an oxytocic 
substance in renal vein blood increased. To de- 



NATIONAL HEART INSTITUTE 



103 



termine the nature of this oxytocic principle it 
has been necessary to determine the specificity 
of the method developed in this laboratory for 
isolating kallidin from blood. Essentially it has 
been found that the method isolates both the 
kallidins and angiotensin but does not isolate 
acetylcholine or serotonin. Chymotrypsin in- 
activates both polypeptides while trypsin, 
under controlled conditions specifically de- 
stroys angiotensin and kallidinase specifically 
destroys the kallidins. Presently our studies 
are directed towards determining the nature of 
the oxytocic principle found in blood and to de- 
termine the nature of increased level of oxyto- 
cic principle found in renal vein blood during 
the infusion of acetylcholine. 

Chronic Electrical Stimulation of the Heart 

The use of electronic stimuli to control the 
heart rate chronically has become a practical 
reality. Recently the use of paired stimuli to 
produce augmented myocardial contractility 
has demonstrated again the need for precise 
analysis of the parameters associated with 
myocardial stimulation. Analogies taken from 
work done with nerve stimulation are no 
longer satisfactory. 

Stimulation energy thresholds are deter- 
mined from the general formula : 

Energy = (voltage) (current) (time) 

To study these thresholds comprehensively 
both acute and chronic electrode implantations 
were made in dogs, using electrodes of known 
size and configuration. The available surface 
area was .055 sq. cm. Acute energy-duration 
curves demonstrated a minimum of approxi- 
mately 0.2 microjoules at pulse durations be- 
tween 0.5 and 1.0 msec. At shorter pulses both 
voltage and current increase rapidly thereby 
rapidly increasing energy thresholds and at 
longer pulses the increased time base increases 
the energy thresholds, though less abruptly, 
demonstrated a very similar curve configura- 
tion, with a minimum near 3.5 microjoules be- 
tween 0.5 and 1.0 milliseconds. 

Energy thresholds have also been shown to 
vary with electrode surface area. If the anode 
surface is enlarged, the total electrode voltage 
Chronic studies (3 m.onths or more) have 



is reduced and current thresholds remain con- 
stant. If the cathode surface area is enlarged 
total voltage also drops (at the same current) 
but current thresholds are increased. When the 
current is increased to stimulation threshold 
levels, the energy threshold has not decreased. 
Thus to minimize the energy required for 
stimulation with these electrodes a small cath- 
ode and large anode should be used with a sti- 
ulus pulse width of 0.5 to 1"0 msec. Further 
study to analyze the electrochemical processes 
which lead to cellular depolarization are in 
progress. 

LABORATORY OF KIDIVEY AND ELEC- 
TROLYTE METABOLISM 

The Laboratory of Kidney and Electrolyte 
Metabolism is devoted to studies of electrolyte 
and water transport across biological mem- 
branes, the role of the renin-angiotensin sys- 
tems in experimental situations associated with 
fluid retention and in hypertension, and to car- 
diac muscle contractility as influenced by a 
protein system present in mammalian plasma. 

Micropuncture Studies in the Kidney 

The importance of micropuncture studies in 
the intact nephron as a means of direct ex- 
amination of renal transport mechanisms is 
self-evident. In a previous report a method for 
pursuing such studies in the proximal nephron 
of the dog was described. Since that time the 
method has been improved significantly, and it 
is now possible to remove repeated samples of 
tubular fluid from the same proximal tubule 
throughout the course of a single study. In the 
past year this technique has been applied to a 
study of the fractional reabsorption of water 
(and by inference of sodium since these are 
reabsorbed by a linked mechanism) in the 
proximal nephron. It had been postulated in 
the past on the basis of indirect evidence that 
fractional reabsorption of tubular fluid in the 
proximal nephron is relatively constant, unin- 
fluenced by changes in glomerular filtration 
rate, but is responsive to the action of various 
diuretic agents. It is known that the injection 
of saline into intact dogs reduces fractional so- 
dium reabsorption by an unknown mechanism 
presumably humoral. On the basis of experi- 



104 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ments performed in this laboratory utilizing 
micropuncture technique, it has been concluded 
in agreement with the earlier inferences that 
fractional reabsorption of water (and sodium) 
is in fact virtually constant in the normal ani- 
mal, and that changes in glomerular filtration 
rate, whether spontaneous or experimentally 
induced, result in rapid adjustment, the net 
effect of which is the maintenance of the con- 
stancy of proximal reabsorption. An elucida- 
tion of the mechanism of this glomerulotubular 
readjustment is an important problem which is 
being actively pursued at the present time. 

It has also been observed that the decrease in 
fractional reabsorption of sodium observed in 
the intact dog following infusion of isotonic or 
hypertonic sodium chloride is a result of a sig- 
nificant decrease in reabsorption in the proxi- 
mal nephron. This firmly establishes the locus 
of the effect of sodium chloride in this respect. 
It is of interest that even at the diminished 
level of sodium reabsorption observed follow- 
intravenous saline, acute alterations in GFR, 
as in the normal animal, do not affect the new 
steady state fractional reabsorptive rate. In 
acute thoracic inferior vena caval obstruction, 
a situation which leads to sodium retention and 
edema accumulation, micropuncture studies of 
the proximal nephron were indicative of an in- 
crease in the rate of reabsorption of fluid in 
this segment. No such effect on proximal reab- 
sorption was noted when venous pressure was 
elevated by obstruction of the abdominal vena 
cava, a procedure which does not generally lead 
to the accumulation of edema. 

On the basis of preliminary studies, it ap- 
pears probable that none of the following di- 
uretic agents: hydrochlorothiazide, chlormero- 
drin, ethacrynic acid, and f urosemide diminishes 
proximal reabsorption. Their effects on sodium 
reabsorption must, therefore, be exerted at a 
more distal site in the nephron. In contrast, 
the infusion of an osmotically active agent, such 
as mannitol, clearly diminishes proximal re- 
absorption as had been assumed on the basis 
of classical clearance studies in the past. 

In collaboration with a visiting scientist 
from Japan, a technique for micropuncture of 
the otherwise inaccessible intrarenal papilla of 
the rat was developed. This required operative 



removal of a portion of the overlying renal cor- 
tex, visualization and mobilization of the intra- 
renal papilla, and subsequent direct micropunc- 
ture of collecting tubules and other structures 
in this segment. Despite the operative inter- 
vention and resultant reduction in renal mass, 
the kidney functions in a qualitatively rela- 
tively normal manner in that the elaboration of 
the hypertonic urine (less so than from the un- 
operated kidney) was observed. Furthermore, 
was possible to establish that, as in species in 
which the papilla was not intrarenal and there- 
fore accessible to direct examination, the osmol- 
alitjr of the tubule fluid in collecting ducts in- 
creases progressively towards the tip of the 
papilla. Finally, a small potential difference of 
11 microvolts, tubule lumen negative to inter- 
stitium, was also observed. 

Isolated Tubules in Vitro 

For a number of years this laboratory has 
been engaged in an analysis of electrolyte ex- 
change within cortical slices of rabbit kidneys 
utilizing classical isotope techniques. The diffi- 
culty of interpretation of the results inherent 
in the heterogeneity of the tissue and the in- 
fluence of non-cellular compartments led to de- 
velopment of a technique to perform similar 
studies in suspensions of isolated cortical tu- 
bules in vitro. The results of these studies have 
been reported in the past and, though the re- 
moval of an interfering extracellular phase in 
the newer preparation minimized some of the 
theoretical difficulties in interpretation, it was 
still impossible to relate the observations to 
transcellular fluxes directly for a number of 
reasons, including the heterogeneity of the tu- 
bule fragments within the suspension. In the 
past year a major improvement in technique 
has been developed which permits the perfu- 
sion of single isolated tubule fragments in 
vitro. The fragments are dissected by hand 
from fresh rabbit kidneys without prior treat- 
ment. It was possible to analyze the water and 
electrolyte content of fragments of glomerulus, 
proximal convoluted tubule, proximal straight 
tubule, thick ascending tubule and collecting 
tubule, and it was observed that the potassium 
content of these segments closely approximates 
that observed in the past in both kidney slices 



NATIONAL HEAET INSTITUTE 



105 



and suspension of renal cortex, evidence, in 
part, that the tubules are viable since they are 
capable of maintaining relatively normal con- 
centration gradients for potassium. Similarly, 
the content of sodium and water of the proxi- 
mal tubule approximates that in slices and sus- 
pensions. The sodium content of the other seg- 
ments has not yet been estimated with certain- 
ty. Isotopic exchange utilizing K*- and NA=- has 
also been performed. 

In the studies alluded to above, and reported 
in a previous Annual Report, a minimum of 
two tissue compartments of each ion had been 
observed in the suspension. It appears likely, 
however, on the basis of the present studies, 
that, insofar as potassium is concerned, these 
compartments may represent different tubule 
segments rather than compartments within the 
same cell. On the other hand, the kinetic com- 
partmentalization of sodium observed in the 
tubule suspension may, in fact, be due to intra- 
cellular heterogeneity, though this has not yet 
been established with certainty. However, at 
least two compartments, one rapid, the other 
slow, have been observed in proximal isolated 
tubule. Since the former is uninfluenced by ex- 
ternal sodium concentration exchange diffusion 
may be excluded ( see below) . 

Having established that such isolated tubules 
are capable of maintaining essentially normal 
electrochemical gradients for sodium and po- 
tassium, micro-perfusion of isolated tubules 
was performed. This required utilization of a 
specially developed concentric double-barreled 
micropipet. It has been established that the 
proximal tubule accumulates chlorphenol red 
as does the intact nephron in situ. Further- 
more, PAH, as in the living kidney, is actively 
transported from an outside bathing medium 
to a PAH-free perfusing fluid (the luminal 
fluid). Further evidence of the adequacy of the 
technique, as well as the viability of the prepa- 
ration, is a demonstration of its impermeabil- 
ity to inulin and its ability to concentrate inu- 
lin within the lumen in consequence of net 
water reabsorption across the cell. Thus, it is 
now possible to evaluate agents which alter 
water permeability of the nephron. A direct 
effect of vasopressin on water permeability 
was observed in perfused segments of collect- 
ing tubule. Both vasopressin and its presumed 



intracellular mediator, cyclic AMP, increased 
the water permeability of the collecting tubule 
as estimated by the use of tritiated water. Vas- 
opressin also increases the net movement of 
water along an osmotic gradient as is assumed 
to occur in the intact kidney. 

Chloride fluxes in both separated renal tu- 
bules and isolated proximal convoluted tubules 
have also been studied. It has been observed in 
the suspension studies that the rate of chloride 
exchange is significantly less than that of so- 
dium, presumptive evidence that earlier esti- 
mates of transcellular transport of sodium 
chloride reported from this laboratory were er- 
roneously high. They were originally based 
only on the very rapid rate of sodium exchange 
noted at that time. 

In neither suspensions of tubules nor isolat- 
ed proximal tubules is there evidence for a 
purely passive distribution of chloride. For ex- 
ample, although the addition of the cardiac 
aglycone strophanthidin, a supposed inhibitor 
of active sodium transport, results in an in- 
crease in the tissue chloride content in the iso- 
lated tubule and in the suspension, no signifi- 
cant effect on cell chloride content is exerted by 
alterations in the concentration of extracellular 
potassium. Thus, the calculated Donnan ratios 
for potassium and chloride differ at all medium 
potassium concentrations, a situation unlike 
that in skeletal muscle in which Donnan equi- 
librium is maintained at all but the lowest ex- 
nal potassium concentrations. 

Toad Bladder 

It had been proposed in a previous report 
that vasopressin increases the permeability of 
the toad bladder and renal tubule to water by 
stimulating the production of an istracellular 
intermediate, cyclic 3', 5'-adenosine monophos- 
phate, in the tissue. The thesis was based on 
physiological studies summarized in detail in 
the earlier reports in which vasopressin, cyclic- 
AMP, and theophylline, which prevents the 
degradation of cyclic-AMP, have similar effects 
on sodium and water movement in toad 
bladder. Direct evidence in support of the 
thesis is provided this year by the demonstra- 
tion that the concentration of cyclic-AMP in 
bladder tissue is significantly increased by 



106 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



preincubation of toad bladder with either vaso- 
pressin or theophylline or a combination of 
both agents. The mechanism of action of cyclic- 
AMP on the biochemical processes within the 
cell as well as the nature of the alteration of 
the structure of the membrane which permits 
more rapid water flow and sodium transport is 
currently under investigation. 

An attempt at characterization of the effect 
of vasopressin and its intermediate (cyclic- 
AMP) on biochemical processes in the toad 
bladder has been initiated. Earlier studies in 
this laboratory had demonstrated that vaso- 
pressin and cyclic-AMP stimulate oxygen con- 
sumption and glycogen utilization by the toad 
bladder incubated in Ringer solution contain- 
ing sodium. No effect on oxygen consumption 
was observed when choline was substituted for 
the sodium in the Ringer solution. In addition 
it was noted that vasopressin or cyclic-AMP 
stimulates phosphorylase activity in toad 
bladder regardless of the sodium content of the 
Ringer solution. In contrast to the results of 
other workers, it has also been shown that a 
number of metabolic inhibitors, including dini- 
trophenol nitrogen, iodacetic acid, etc. inter- 
fere with the effects of vasopressin on water 
and sodium movement across the toad bladder, 
whereas certain other inhibitors do not. The 
results thus far suggest that basal sodium 
transport and stimulation of sodium transport 
by vasopressin are not dependent on an intact 
glycolytic pathway but require an intact tricar- 
boxylic acid cycle. On the other hand, the effect 
of the hormone on water movement does not 
appear to be closely linked either to glycolysis 
or to the tricarboxylic acid cycle. It appears to 
be intact if either pathway is available. This is 
based in part on the observation that a specific 
inhibitor of the Krebs cycle which limits the 
sodium response does not interfere with the 
water response to hormone, whereas the inhibi- 
tory effect on the water response of at least one 
of the glycolytic inhibitors can be reversed by 
the addition of substrate, namely pyruvate. 

A more precise approach to the metabolic 
effect of vasopressin has been undertaken by 
measuring the effects of the hormone on the 
pool sizes of various biochemical intermediates 
and co-factors in bladder. Thus far it has been 
observed that vasopressin increases the concen- 



tration of glucose-6-phosphate and certain 
other glycolytic intermediates; this correlates 
well with the results of studies on phosphory- 
lase activation mentioned earlier. No change 
was observed in the concentration of adenosine 
triphosphate and adenosine diphosphate. 

The effect of a naturally occurring fatty acid, 
prostaglandin, which has been isolated by others 
from a number of sources including human 
vesicular plasma, on water permeability of the 
toad bladder has been examined. Prostaglan 
while itself without effect on permeability, in 
extremely low concentrations, is capable of sig- 
nificantly inhibiting the permeability response 
to antidiuretic hormone and theophylline, but 
not to that of cyclic-AMP. Whether this indi- 
cates a physiological regulatory role of prostag- 
landin on water movement in vivo is unknown. 

The influence of changes in the cation com- 
position of the bathing medium and of the toad 
bladder cell itself on vasopressin responsive- 
ness is also under study. Thus far it is apparent 
that removal of potassium from the blood sur- 
face of the toad bladder causes a marked de- 
pression of sodium transport and the effect of 
vasopressin on sodium transport within a rela- 
tively short time. In contrast, the effect of the 
hormone on water permeability is not altered 
until a considerably longer period, approxi- 
mately one hour, has elapsed. The diminished 
permeability response with respect to water 
appears to be best correlated with a decrease 
in the cell content of potassium. Thus, though 
strophanthidin, a cardiac aglycone, inhibits so- 
dium transport immediately in toad bladder, it 
does not interfere with the water response to 
ADH until a significant decrease in potassium 
content of the tissue develops. This effect is 
rapidly reversible upon addition of potassium 
to the bathing medium. 

Finally, in an effort of dissociate the effect of 
vasopressin and its analogues on water and so- 
dium movement, the effects of these compounds 
in the same bladder on simultaneously deter- 
mined net water movement and short circuit 
current (an estimate of net sodium transport) 
were examined. It was not possible to define 
clearly any important differences between the 
various analogues studied with a single excep- 
tion. Arginine vasotocin appeared to yield less 



NATIONAL HEART INSTITUTE 



107 



stimulation of sodium transport than enhance- 
ment of water permeability at a dose at which 
other compounds yielded equivalent effects on 
the two functions. 

Electrolyte Transport Across Red Cells 

The studies of the characteristics of electro- 
lyte transport across red cell membranes of a 
variety of species are continuing. As in the 
past, hemolyzed preparations of red cells (so- 
called ghosts) in which it is possible to vary 
the intracellular invironment at will, have been 
used to advantage. On the basis of earlier work 
in this and other laboratories, it had been as- 
sumed that transport of ions, as measured by 
unidirectional fluxes, can be operationally sepa- 
rated into at least three distinct components; 
active transport, that is, transport against an 
electrochemical gradient, passive diffusion, 
movement downhill along the electrochem- 
ical gradient, and so-called exchange dif- 
fusion. This last is thought to be a one to one 
exchange of an ion — for example, sodium for 
sodium or potassium for potassium. Exchange 
diffusion in which no net movement can be ac- 
complished is experimentally defined by meas- 
uring the change in the unidirectional flux of 
an ion as a function of the concentration of 
that ion in the receiving medium. Thus, that 
component of sodium efflux, for example, which 
is eliminated when sodium is removed from the 
external medium, is said to represent exchange 
diffusion. 

Recent studies have cast considerable doubt 
on this operational definition. Since exchange 
diffusion is symmetrical, any manipulation 
which modifies one of the unidirectional fluxes 
should exert a similar effect on the other. In 
many situations this has not turned out to be 
the case. Thus, although a decrease in external 
sodium diminishes sodium efflux, a predictable 
change in sodium influx does not occur in asso- 
ciation with changes in the intracellular so- 
dium content. The sodium sensitive efflux also 
appears to be energy dependent. Cells depleted 
of metabolic substrate lose the ability to pump 
sodium and potassium but still retain the so- 
dium-sensitive component of efflux; this too 
disappears after further metabolic depletion. 
Also, a differential effect of substrate on the 



active transport process and the sodium sensi- 
tive process has been discerned. The addition 
of inosine to cells devoid of both processes acti- 
vates the sodium-sensitive component of efflux, 
but does not affect that dependent on external 
potassium. In contrast, adenosine reactivates 
both forms of transport. 

Studies of potassium efflux have similarly 
shown that the fraction of efflux dependent on 
external potassium does not satisfy the criteria 
for exchange diffusion. Thus, glycosides inhibit 
the potassium-sensitive efflux without a 
symmetrical inhibition of potassium influx. 
The two nucleosides, adenosine or inosine, in- 
hibit the potassium-sensitive efflux but have no 
effect on influx. It is therefore clear that the 
operational definition of sodium and potassium 
exchange diffusion as those components of flux 
dependent on the trans concentration of the ca- 
tion requires revision. The sodium-sensitive 
efflux may represent a form of active sodium 
transport which does not require potassium. 

One interesting aspect of the problem has 
been disclosed in a study of red cells from pa- 
tients with cystic fibrosis, since the electrolyte 
and water composition of these cells is indis- 
tinguishable from that of normal ones. No po- 
tassium-sensitive potassium efflux component 
is discernible. 

Dog red cells which differ from human and 
many other animal red cells in that they con- 
tain a high rather than low concentration of 
sodium and a low rather than high concentra- 
tion of potassium have also been studied. These 
cells appear to lack a cardiac glycoside-sensi- 
tive cation pump. Furthermore, the permeabil- 
ity to both sodium and potassium are remark- 
ably sensitive to changes in cell volume. 
Shrunken cells are highly permeable to sodium 
and relatively impermeable to potassium, 
whereas when swollen these same cells have a 
low permeability to sodium and a high permea- 
bility to potassium. These effects are also meta- 
bolically linked in that energy-depleted cells do 
not reveal the volume-induced changes in per- 
meability unless additional substrate is pro- 
vided. The steroid, aldosterone, in confirmation 
of work reported by others, retards the influx 
of Na=^ into dog red cells — an effect which is 
demonstrable only in the presence of plasma, 



108 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



indicative of the necessity of some plasma co- 
factor for the phenomenon. 

The relationship between the rates of glycol- 
ysis and cation transport in human red cells 
has also been re-examined. In the past it had 
been observed that glycosides which interfere 
with or inhibit active transport do not simul- 
taneously alter the steady state production of 
lactate, indicative of an absence of effect on 
glycolysis. This observation was made in utiliz- 
ing normal red cells. In contrast, stored cells, 
in which some degree of substrate depletion is 
achieved, respond to glycosides by a further re- 
duction in lactate production. On the basis of 
preliminary observations, it appears that this 
effect is related to the altered intracellular so- 
dium and potassium content, and that sodium 
transport and lactate production are linked. 
Thus, high sodium cells under certain condi- 
tions transport sodium more rapidly and simul- 
taneously increase lactate production. The 
effect is analogous to that noted in tissues such 
as kidney and frog skin in which the level of 
oxygen consumption appears to be regulated by 
the rate of sodium transport. 

Renin-Angiotensin Aldosterone System 

An important segment of the laboratory is 
devoted to an intensive and long-term study of 
the role of the renin-angiotensin-aldosterone 
system in fluid accumulation (experimental 
heart failure) and hypertension. In the past 
they have provided important evidence that the 
renin-angiotensin system in the kidney is in- 
volved in the regulation of aldosterone secre- 
tion by the adrenal. Further evidence has been 
accumulated in the past year concerning the 
role of this system in congestive heart failure, 
hypertension and other experimental states. 

Hypertensive dogs have been studied in an 
effort to determine the relationship of plasma 
renin concentration and aldosterone secretion 
to blood pressure. For this study two groups of 
experimental animals, one with acute experi- 
mental renal hypertension, the other with 
chronic experimental renal hypertension, has 
been compared to normal controls. It was noted 
that an elevation of plasma renin and increased 
rate of aldosterone secretion occurred only 
during the first few days of hypertension in an- 



imals which ultimately developed the chronic 
form of the disease. In contrast, in dogs with 
acute malignant hypertension plasma renin re- 
mained elevated throughout the entire course 
of the disease which lasted for as long as 13 
days. In these, aldosterone secretion was also 
generally elevated. Arterial hypertension de- 
veloped in both groups approximately two day 
following application of a Goldblatt clamp to 
the renal artery. 

In another study directed at evaluating the 
role of renin hyperaldosteronism in renal hy- 
pertension, an attempt was made to determine 
whether renal artery constriction would, as in 
normal animals, produce hypertension and a 
further increase in plasma renin in animals 
with experimental secondary hyperaldosteron- 
ism. The animals in the control phase of the 
study all had increased plasma renin content 
and hyperaldosteronism, but of 16 dogs with 
secondary aldosteronism due to thoracic caval 
constriction, only 4 developed hypertension 
following renal artery clamping, and plasma 
renin rose to higher levels in only 2 of these ani- 
mals. Of 6 sodium-depleted animals with secon- 
dary hyperaldosteronism, in only 2 did hyper- 
tension develop following clamping and in none 
did plasma renin increase. Sodium depletion of 
5 dogs with renal hypertension, on the other 
hand, produced a "normal" response in that 
plasma renin increased. 

Sodium depletion is known to result in en- 
hanced rates of aldosterone secretion and in- 
creased plasma renin concentration. However, 
in addition, evidence has accumulated to indi- 
cate that an associate increase in corticoster- 
one secretion does not take place. This appears 
to be a consequence of an inhibitory effect of 
the intact pituitary since both aldosterone 
secretion and corticosteroid secretion are aug- 
mented following sodium depletion in hypophy- 
sectomized animals. The enhancement in aldo- 
sterone secretion is not as great under these 
conditions as in non-hypophysectomized dogs 
indicative of an important supportive role of 
the anterior pituitary. 

A delay in the metabolism of aldosterone 
may play a significant role in certain situations 
associated with hj'-peraldosteronism. In order 
to evaluate this possibility, the disappearance 
of tritiated aldosterone from peripheral plasma 



m. 



NATIONAL HEART INSTITUTE 



109 



was studied in a number of experimental con- 
ditions. It was noted that the slow component 
of the disappearance curve was prolonged and 
the so-called metabolic clearance rate dimin- 
ished in dogs after hemorrhage, following hy- 
pophysectomy, and in experimentally induced 
low output heart failure. Aldosterone metabo- 
lism, on the other hand, was not detectably al- 
tered in dogs with high output failure or 
during sodium depletion. It is assumed, there- 
fore, that hyperaldosteronism seen in the first 
three conditions is in part at least a conse- 
quence of the decrease in the rate of the degra- 
dative process. Since there is negligible extra- 
hepatic clearance of aldosterone from plasma, 
it was of interest to study the characteristics 
of hepatic extraction of the steroid in a series 
of animals. It was concluded that the hepatic 
mechanism for the metabolism of aldosterone 
is flow limited rather than limited by the rate 
of hepatic metabolism, since virtually complete 
extraction of aldosterone was accomplished by 
the liver in normal dogs as well as in those fol- 
lowing hemorrhage, sodium depletion, hypo- 
physectomy, and during severe secondary con- 
gestion of the liver due to thoracic caval con- 
striction. Finally, it was observed that hepatic 
blood flow is diminished in those situations in 
which aldosterone metabolism is delayed. 

One of the more puzzling problems relating 
to aldosterone is the increased sensitivity of 
the kidney to salt-retaining adrenal steroids in 
certain states associated with experimental or 
spontaneous fluid retention. Thus, the animal 
with a constriction of the thoracic vena cava is 
exquisitely sensitive to desoxycorticosterone 
and the salt retention produced by this agent 
persists throughout the course of a prolonged 
period of administration. In contrast, the nor- 
mal dog, as does man, accumulates fluid only 
transiently and despite continued administra- 
tion of large doses of desoxycorticosterone, ap- 
pears to "escape" from the salt retention 
effects of the hormone. This laboratory has 
suggested in the past that the escape phenom- 
enon may be regulated by, or dependent on, 
another circulating hormone. In an effort to de- 
termine the validity of this thesis, first by ex- 
clusion and then by direct demonstration, the 
following studies were performed: The role of 
a decrease in glomerular filtration rate in the 



development of the increased sensitivity of the 
renal tubules to the salt-retaining steroid was 
eliminated by demonstrating that a "normal" 
escape from desoxycorticosterone occurs in un- 
ilaterally nephrectomized animals in which fil- 
tration rate is reduced by application of a renal 
arterial clamp. 

Of greater significance has been the recent 
observation that cross-transfusion of blood 
from "DOCA-escape" sodium-loaded dogs to 
normal recipient animals resulted, in six suc- 
cessful studies, in the development of some de- 
gree of natriuresis in the recipient animal. Al- 
though an increase in filtration rate and renal 
blood flow in the recipient animal usually oc- 
curred, this was not a uniform finding and, in 
some there was either no change or a fall in 
GFR. Of significance is the fact that natri- 
uresis occurred in all regardless of the changes 
in filtration rate. It seems likely, therefore, 
that a humoral factor may have been respon- 
sible for these results, although this cannot be 
concluded with certainty as yet. 

The biochemical synthesis of aldosterone is 
thought to involve desoxycorticosterone as a 
precursor for corticosterone, the latter, in turn 
preceding aldosterone in the synthetic chain. It 
is of interest, therefore, that the infusion of 
angiotensin, a known stimulator of aldosterone 
and corticosterone secretion, did not increase 
the secretion of desoxycorticosterone from the 
adrenal gland of the dog. Similarly, desoxycor- 
ticosterone secretion is not increased in dogs 
with experimental congestive heart failure, 
though the secretion of aldosterone and corti- 
costerone are. 

The effects of the administration of the diur- 
etics, organic mercury and chlorothiazide, on 
plasma renin and aldosterone secretion were 
also studied. Following diuresis due to both of 
these compounds, plasma renin and aldosterone 
secretion increased. In the case of chlorothia- 
zide the augmentation of plasma renin and al- 
dosterone was prevented by administration of 
intravenous saline at a rate in excess of the 
drug-induced sodium loss. It was concluded in 
agreement with other investigators that renin 
release and subsequent enhancement of aldos- 
terone secretion following chlorothiazide are 
the consequences of prior salt loss and not a 
direct effect of the compound. Although similar 



110 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



conclusions may apply to the effect of mercury, 
the results of replacement of sodium were not 
uniform, and in some dogs this did not prevent 
the renin and aldosterone changes. 

Finally, the renin-angiotensin system has 
been examined in other animals. On the basis 
of preliminary results, it appears that a system 
which functions in a manner similar to that in 
the dog and in other mammals is present in the 
very primitive opossum. Similar studies are in 
progress in the American bullfrog. In the 
white laboratory rat the principal glucocorti- 
coid secreted by the adrenal is corticosterone ; 
this is not the case in the golden hamster. In 
the latter Cortisol is the major glucocorticoid 
present in adrenal blood, and furthermore, 
detectable amounts of aldosterone and corticos- 
terone are also present. 

Cardiac Muscle Contraction 

It has been previously reported that cardio- 
globulin-C, one of the three fractions of the 
inotropic cardioglobulin system, contains cal- 
cium. It was also suggested that cardioglobu- 
lin-C may, in part, act as a carrier for the cal- 
cium necessary for the induction of shortening 
of muscle contractile protein. In the past year 
it has been possible to determine with consider- 
able accuracy the calcium content of ashed car- 
dioglobulin-C samples. On the basis of an 
analysis of the amount of cardioglobulin-C ne- 
cessary for a maximal inotropic effect in frog 
heart, the amount of cardioglobulin calcium 
bound to frog heart under these circumstances 
has been calculated. This figure has been com- 
pared to a similar estimate based on radioac- 
tion induced by cardioglobulin in the absence of 
external calcium involves the movement of car- 
consistent with the view that muscle contrac- 
induced by cardioglobulin in the absence of ex- 
ternal calcium involves the movement of car- 
dioglobulin-C calcium in an amount which is 
comparable to the influx of free calcium report- 
ed by others to be associated with a single frog 
heart contraction. 

In the earlier report the results of cardioglo- 
bulin assays in the plasma of patients with so- 
called idiopathic heart failure have been sum- 
marized. Approximately 50% of these patients 



had abnormally low cardioglobulin content, and 
this was considered to reflect either a diminu- 
tion in cardioglobulin-C or cardioglobulin-A. 
Utilizing a more precise assay system devel- 
oped in the past year this difference between 
the control and experimental groups with idio- 
pathic heart failure was not observed. This re- 
sult has stimulated a reinvestigation of the en- 
tire problem. 

If the cardioglobulin system is a naturally 
occurring system in the mammal and its bind- 
ing characteristics to heart muscle are similar 
to that observed in the isolated frog heart, it 
should be possible to demonstrate the localiza- 
tion of cardioglobulin-C on the surface of ma- 
malian heart muscle. With this in mind, an 
immunofluorescent antibody study has been ini- 
tiated. Antibodies to rat cardioglobulin-C were 
induced in the guinea pig and were then added 
to a number of tissues from the rat. Localiza- 
tion was determined by standard immunofluo- 
rescence techniques. It was assumed that locali- 
zation would indicate the site of endogenous 
cardioglobulin-C in the rat. That antibody to 
cardioglobulin-C was in fact formed in the 
guinea pig was proved by the ability of the 
guinea pig antiserum to inhibit the inotropic 
effect of cardioglobulin-C on frog heart as well 
as the demonstration of its binding to cardio- 
globulin-treated heart, but not to untreated 
heart. Although antisera did in fact "stick" to 
rat heart surface, it also adhered to skeletal 
muscle and to a number of other tissue sites 
including basement membranes of the renal 
glomerulus, renal tubules, etc. Binding could be 
prevented by preliminary passage of the anti- 
sera through cardioglobulin-treated frog heart 
as well as by exposure to washed homogenized 
rat kidney. 

The appropriate interpretation of these find- 
ings is not yet clear. 

LABORATORY OF METABOLISM 

The work of the Laboratory of Metabolism 
will be summarized here by Section. Members 
of the three Sections continue to collaborate 
productively on a number of problems, but the 
bulk of the work in each group stands inde- 
pendently and is so reviewed. 



NATIONAL HEART INSTITUTE 



111 



Section on Metabolism 

Mobilization and Utilization of Free Fatty 
Acids 

Metabolism of Adipose Tissue Studied In 
Vitro. The first direct evidence in cell-free 
preparations for the presence of two independ- 
ent systems for activating and for inactivat- 
ing adipose tissue lipase, respectively, has been 
obtained. Demonstration of this was made pos- 
sible by a large difference in pH optimum for 
the two processes. The activating system was 
prepared in a particle-free fraction and some 
of its properties were determined. Through the 
rapid operation of these systems, it is now 
clear that the adipose tissue can rapidly adjust 
its rate of mobilization of free fatty acids 
(FFA) by inter-converting active and inactive 
forms of the hormone-sensitive lipase. The stud- 
ies in cellfree preparations establish that the 
process is not dependent upon synthesis of new 
enzyme protein but, like the activation of phos- 
phorylase, represents conversion of an inactive 
to an active form of the enzyme. 

The Cortisol stimulation of adipose tissue lip- 
ase activity has been confirmed. F urther stud- 
ies show that the use of serum as an incuba- 
tion medium strikingly enhances the Cortisol 
effect. Dialyzed serum on the other hand is 
much less effective, but simple addition of glu- 
cose restores its effectiveness. It is concluded 
that maximum Cortisol stimulation requires 
both a non-dialyzable fraction of serum and 
glucose. It is hoped that further studies along 
this line, since they relate to a specific and 
readily investigated system (i.e. activation of 
lipase), may increase our understanding of the 
still mysterious mechanism of action of Corti- 
sol. 

Techniques for study of adipose tissue with 
the electron imcroscope are being developed. 
Earlier studies in this laboratory showed the 
presence of very small, electron-dense particles 
in homogenates of adipose tissue. These par- 
ticles have now been demonstrated in the cyto- 
plasm of intact fat cells, and preliminary stud- 
ies indicate that the distribution of these is 
altered by exposure of the tissue to lipolytic 
hormones. These particles are isolated along 
with the fat layer at the top of the tube after 
centrifugation of homogenates of adipose tis- 



sue. This layer contains most of the lipase acti- 
vity of the cell and also most of the proteolytic 
activity involved in the degradation of peptide 
hormones. Further studies are planned to de- 
termine whether these enzymatic activities ac- 
tually reside in the described particles. 

Effects of Prostaglandin on FFA 
Mobilization. It was first shown in this 
laboratory last year that prostaglandin Ei 
PGEi) is remarkably potent, both in vitro and 
in vivo, in counteracting the fat-mobilizing ac- 
tion of catecholamines. Concentrations as low as 
10"^ M in vitro are effective in counteracting 
stimulation of FFA release by catecholamines 
present in 2 to 5 times this molar concentration. 
It has not been shown that in unanesthetized 
dogs, PGEi can profoundly depress plasma FF 
levels when given in single large doses intra- 
venously. Investigators at the Karolinska Insti- 
tute in Stockholm have observed increases in 
plasma FFA levels in man during constant in- 
travenous infusions of PGEi. Unanesthetized 
dogs do not show this response under the same 
experimental conditions. It will be of interest 
to determine whether or not human adipose 
tissue in vitro responds differently from rat 
adipose tissue. 

The findings in these PGEi studies are diffi- 
cult to reconcile with the Handle hypothesis 
regarding competition between FFA and glu- 
cose for utilization by peripheral tissues. PGEi 
caused profound drops in FFA level with 
either no change or a slight increase in plasma 
glucose level. Similar observations have been 
made in man during intravenous administra- 
tion of nicotinic acid. If high levels of plasma 
FFA suppress glucose utilization, one might 
expect the plasma glucose levels to drop when 
the plasma FFA level is rapidly dropped to 
very low levels. Definitive conclusions will re- 
quire measurement of glucose turnover using 
radioisotopes. 

UTILAZATION of FFA BY ISOLATED CELL 

Suspension. New insights into the basic mech- 
anisms and the kinetics of FFA utilization 
have been obtained. The Ehrlich ascites tumor 
cell was chosen as a readily available model 
system for studying isolated individual cells 
with regard to FFA uptake and utilization. As 
reported last year, the initial transfer of FFA 



112 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



from FFA-albumin complexes to the cell sur- 
face takes place with remarkable rapidity. The 
mechanism appears to depend upon prior disso- 
ciation of the complex and then uptake of the 
FFA anions. It has now been shown that the 
subsequent utilization, both for oxidation and 
esterification, occurs at a rate that correlates 
with the total "load" absorbed initially onto the 
cells. The latter is shown to be a function of the 
molar ratio of FFA to albumin in the medium 
to which the cells are exposed. From these stu- 
ies, it is now clear that overall FFA utiliza- 
tion will be dependent ultimately on this molar 
radio in the bathing medium. These results in 
the model system are consistent with a number 
of observations made in perfused mammalian 
tissues and in tissue slices. 

The binding sites on the cell do not appear to 
be specifically "assigned" to individual long- 
chain fatty acids. The total binding of FFA is 
approximately the same whether a single spe- 
cies of fatty acid is used or whether a mixture 
of different fatty acids is used. If these results 
can be extrapolated, they imply that the differ- 
ences in uptake of different fatty acid species 
that have been observed in other studies are to 
be attributed not to differences in binding, but 
rather to differences in Km of the enzymes re- 
sponsible for subsequent metabolic transforma- 
tions of the FFA taken up initially. 

Effects op FFA and op Catecholamines 
ON Myocardial Energy Metabolism. Stud- 
ies reported last year showed for the first 
time that perfusion of the isolated rat heart 
with a medium containing concentrations of 
FFA increases the total oxygen consumption of 
the myocardium. It has now been shown that a 
non-metabolizable fatty acid analogue 
(3,3,12,12-tetramethylmyristic acid) does not 
cause this stimulation of oxygen consumption, 
suggesting that it is not the long-chain fatty 
acid per se, but rather some subsequent metab- 
olite (e.g. fatty acyl CoA) that is the effective 
form. This analogue has been shown to be very 
poorly incorporated into esters, and it is not 
oxidized at all. 

The percentage increment in oxygen con- 
sumption caused by FFA was much smaller in 
the beating heart than it was in the heart ar- 
rested by perfusion with high concentrations 



of potassium. This suggested the possibility 
that the pattern of electron transport supply- 
ing energy for what might be called the "basal 
metabolism" of the myocardium might be dif- 
ferent from that prevailing in the beating 
heart. Recent studies with oligomycin are con- 
sistent with this possibility. Oligomycin rap- 
idly causes degeneration of the beating heart 
with arrest within 5 or 10 minutes. On the 
other hand, oligomycin under the same condi- 
tions does not significantly depress oxygen con- 
sumption of the potassium-arrested heart. 
Even in the presence of oligomycin, FFA stim- 
ulate oxygen consumption of the arrested 
heart. It has been shown in Chance's labora- 
tory that oligomycin blocks ATP formation but 
does not prevent utilization of high energy in- 
termediates for certain cell functions, such as 
ion transport. The present results suggest, 
then, that the "basal metabolism" of the ar- 
rested heart preparation may depend to a large 
extent on reactions not linked obligatorily to 
ATP formation. 

Evidence was obtained to show that in addi- 
tion to its chronotropic and inotropic effects, 
epinephrine stimulates oxygen consumption by 
an independent mechanism in the arrested 
heart preparation. This increase in oxygen con- 
sumption of the arrested heart was accompan- 
ied by an increase in the rate of release of glyc- 
erol to the perfusing medium and an increase 
in the FFA concentrations within the myocar- 
dium. This raises the possibility that the un- 
derlying mechanism may be related to that 
responsible for the increased oxygen consump- 
tion produced by perfusing the heart with a 
medium of high FFA concentration. 

Finally, recent studies show that octanoate 
and ^-hydroxybutyrate stimulate oxygen con- 
sumption in the arrested heart even though 
they do not cause significant stimulation in the 
beating heart. Because the absolute increment 
might be difficult to demonstrate significantly 
in the beating heart, it cannot be concluded 
that the mechanism of the effect is different in 
the two preparations. The importance of this 
observation is that it suggests that there may 
not be a basic difference between long-chain 
and short-chain fatty acids in the system under 
investigation. 



NATIONAL HEART INSTITUTE 



113 



\wt Clinical Studies of FFA and Tri- 
glyceride Turnover. In collaboration with Dr. 
Mones Berman, an intensive study of the kinet- 
ics of FFA turnover and of the utilization of 
FFA for synthesis of plasma triglycerides has 
been undertaken, utilizing computer techniques 
for analysis of results. The results suggest (a) 
that there is a large pool of fatty acids in rapid 
dynamic equilibrium with the plasma FFA, and 
(b) that fatty acids entering the liver are mixed 
with a large pool prior to discharge into the 
plasma compartment again as triglyceride fatty 
acids. From studies of the rate of appearance 
of radioactivity in plasma triglycerides, it is 
possible to estimate the turnover of the latter. 

In normal individuals, the half-life of the to- 
tal low-density lipoprotein triglyceride appears 
to be about 2 hours. The validity of the model 
was tested in a patient with fat-induced hyper- 
lipemia, and here the half -life was found to be 
greater than 20 hours. 

In order to test the validity of this approach 
more directly, a method has been devised for 
the in vitro labeling of human plasma lipopro- 
tein triglycerides. Preliminary animal studies 
suggest that the method for in vitro labeling 
does not alter the native state of the lipopro- 
tein and that the initial rate of removal of lipo- 
protein labeled in this way parallels the rate of 
removal of biologically labeled lipoproteins. 
This method will be used to obtain by direct 
means values for the extent to which triglycer- 
ides return fatty acids to the FFA pool. The 
method promises to be extremely valuable in 
connection with studies of triglyceride turn- 
over in hyperlipemic states. 

Metabolic Studies of Refsum's Syndrome, a 
neiv Lipid Storage Disease 

Heredopathia atactica polyneuritiformis 
(Refsum's syndrome) is characterized by 
retinitis pigmentosa, hypertrophic peripheral 
neuropathy, cerebellar ataxia, elevated levels of 
protein in the spinal fluid, electrocardiographic 
abnormalities and, in some cases, by sudden 
death. The disease is familial and the pattern 
of inheritance suggests a recessive mode. In 
1963, Klenk and Kahlke in Germany first 
reported a remarkable accumulation of phytan- 



ic acid (3,7,11,15-tetramethylhexadecanoic 
acid) in the blood and tissues of these patients : 



CHs 



CHs CHa CHs 

\ I I I 

CHCH2CH2CH!CHCH2CH!CHjCHCH2CHjCH!CH2CHCH2COOH 

CH3 

(Phytanic acid) 

The tetra-terpenoid structure of this acid sug- 
gested that it might be formed by extension of 
the pathway for biosynthesis of cholesterol. 
Addition of a fourth isoprene unit to farnesyl 
pyrophosphate could generate the branched- 
chain carbon skeleton which might be further 
modified to form phytanic acid. The second 
possibility considered was that phytol, a nor- 
mal constituent of the chlorophyll molecule, 
might be absorbed and converted to phytanic 
acid: 



CHs 
\. 



CHs 



CHj 



CH3 



/ 

CHs 



CHCH2CH2CH2CHCHjCH2CH8CHCH:aCH2CHjC=CHCH2CH 



(Phytol) 



Because we were unable to identify a case of 
Refsum's syndrome in the United States, des- 
pite many inquiries at a number of university 
centers, we got in touch with Professor Sigvald 
Refsum in Oslo and established a collaborative 
program with his group at Rikshospitalet. 
Clinical experiments with labeled substrates 
prepared here are being carried out in Oslo, 
and isolation and analysis of metabolites is be- 
ing conducted on samples shipped to us. Con- 
currently, the group in Oslo is carrying out an- 
cillary studies at Rikhospitalet. 

Studies after intravenous injection of meva- 
lonic acid-2-C" failed to reveal significant in- 
corporation into plasma phytanic acid, even 
though incorporation into plasma cholesterol 
occurred at a normal rate. 

Meanwhile, studies carried out here demon- 
strated for the first time that orally adminis- 
tered radioactive phytol is readily absorbed by 
the rat and converted to phytanic acid. By 
feeding large daily doses of phytol (5% in the 
diet) it was possible to produce striking accu- 
mulation of phytanic acid in the plasma and 
liver of the rat. Phytanic acid accounted for 
over 20 % of the total fatty acids in plasma and 
liver. Using C "-phytol, it was shown that the 
compound is well absorbed, primarily by way 
of the lymph, and that over 30% of the ab- 



114 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



sorbed dose was converted to C^Oz in the first 
24 hours. In other words, the normal animal 
has a considerable capacity to metabolize this 
branched-chain structure. This is consistent 
with the fact that very large daily intakes were 
necessary in order to produce accumulation of 
phytanic acid. The amounts of phytol accumu- 
lating were rather small, indicating that phytol 
is very readily converted to the acid by reduc- 
tion of the double bond and oxidation of the 
alcohol function. It was shown that feeding 
phytol to germ-free rats also caused accumula- 
tion of phytanic acid, proving that the conver- 
sion was not dependent upon the action of in- 
testinal micro-organisms. 

With these results in experimental animals 
as a basis, Ci*-phytol was fed to normal con- 
trol subjects in the Clinical Center and to a 
subject with Refsum's syndrome in Oslo. Both 
the normal subject and the patient absorbed 
more than 80% of the tracer dose, The normal 
subject oxidized 27% of the administered dose 
to C^Oa in the first 12 hours. In contrast, the 
patient with Refsum's syndrome oxidized less 
than 3% to C^^O, in the first 12 hours. Both 
the control subject and the patient showed a 
peak of phytanic acid radioactivity in the 
plasma at 6 hours. This disappeared rapidly in 
the normal subject and had virtually all disap- 
peared by the second day, whereas the patient 
showed a second large peak of radioactivity at 
2 days, and this fell only slowly over the next 4 
days. The initial peak presumably reflects the 
presence of labeled phytanic acid in chylomi- 
crons: studies in rats show that the phytol is 
partly converted to phytanic acid during the 
course of absorption, 15% of the label in the 
chylomicrons being present as phytanic acid 
and the remainder as phytol. The second large 
peak in radioactivity in the patient with Ref- 
sum's syndrome presumably reflects secretion 
of phytanic acid into the plasma lipoprotein 
compartment. The extremely slow subsequent 
disappearance and the very poor conversion to 
C"02 strongly suggests that the metabolic er- 
ror in this disease lies in the pathway for oxi- 
dation of the branched -chain structure of phy- 
tol and/or phytanic acid. This conclusion is 
supported by concurrent independent studies at 
Rikshospitalet showing that patients with Ref- 
sum's syndrome have a much lower capacity to 



convert capric acid to its omega oxidation de- 
rivative, sebacic acid. 

A screening program was initiated to deter- 
mine whether phytanic acid or other abnormal 
fatty acids could be found in the plasma of pa- 
tients with neurological diseases similar to 
Refsum's disease. Patient material from a 
number of medical centers around the country 
has been studied, as well as patient material at 
the Naval Medical Center and on the service of 
Dr. Engle and Dr. Carr here at N.I.H. More 
than 20 cases of retinitis pigmentosa have been 
examined, and a few patients with amyo- 
trophic lateral sclerosis, Dejerine-Sottas syn- 
drome, spino-cerebellar degeneration, multiple 
sclerosis, familial dysautonomia, Sjogren-Lars- 
son syndrome, various ataxias and peripheral 
neuropathies have been examined. In none of 
these was phytanic acid or any other abnormal 
fatty acid found. From all of these results taken 
together, it appears that patients with Ref- 
sum's syndrome display a highly specific meta- 
bolic error; that is, the accumulation of phy- 
tanic acid is probably not secondary to the 
nerve degeneration. The isotope studies indi- 
cate that biosynthesis is not a major source, 
but that phytanic acid probably has an exogen- 
ous origin. Phytol is shown to be a very effec- 
tive precursor, but it remains to be established 
whether the dietary intake of phytol itself or 
of other potential precursors is adequate to ex- 
plain the accumulation in these patients. What- 
ever the origin of the phytanic acid, the pres- 
ent results indicate that the metabolic error 
lies in the oxidative pathway. It is quite pos- 
sible that the enzyme defect embarrasses a 
metabolic pathway or pathways in addition to 
that for phytanic acid degradation. Studies are 
in progress to determine the steps involved in 
the breakdown of phytanic acid so that the spe- 
cific site of the block in Refsum's syndrome can 
be pinpointed. 

Studies of Lymphatic Lipids and their Origin 

It has been known for some time that even in 
the fasting animal there is a continuing pro- 
duction of lipids appearing in the lymph 
drained from the thoracic duct. However, the 
origin and nature of this "endogenous lymph 
lipid" has not been previously determined. Us- 




. NATIONAL HEART INSTITUTE 



115 



ing techniques for cannulation developed in 
this laboratory, this problem has been investi- 
gated in rats maintained on a fat-free diet. It 
was shown that most of the lipid appearing in 
the thoracic duct lymph actually has its origin 
in lymphatics draining the small intestine, i.e., 
it does not come primarily from liver lymph. 
The material is mostly contained in very low- 
density lipoproteins floating at density 1.006 in 
the centrifuge. Because the concentration in the 
lymph is higher than that in the plasma, it 
seems unlikely that this is derived from the 
plasma. Studies after injection of radioactive 
fatty acids show that the circulating FFA frac- 
tion is not a major source. Lipids delivered to 
the duodenum with bile may account for about 
half of the endogenous lymph lipids, and the 
remainder presumably synthesized directly in 
the intestinal wall. 

The extent to which the intestine contributes 
lipids to the plasma has never been established 
with certainty. Recent studies in man suggest 
that this may be of significance. The present 
results in the rat, if combined with ancillary 
studies of plasma lipoprotein origins, may be 
of great importance in resolving this problem. 

In conjunction with the studies of C"-phy- 
tol metabolism described above, an intensive 
study of its lymphatic absorption has been car- 
ried out. Although most of the phytol absorbed 
enters by way of the lymph in most animals 
studied, an occasional rat appears to absorb 
phytol to a significant degree by another route. 
This is currently under investigation. Most of 
the radioactivity in the lymph is phytol, but 
this is largely present in a combined form, pos- 
sibly long-chain fatty acid esters of phytol. 
About 15% of the radioactivity is found in 
phytanic acid, indicating conversion of the al- 
cohol to the acid in the intestinal wall. The 
phytanic acid also appears to be present largely 
in combined forms. 

Studies of Membrane Lipoproteins 

Little is known about the basic structure of 
membrane lipoproteins. These proteins are 
only sparingly soluble in ordinary media, and 
this has made difficult the fractionation and 
characterization of them. Some progress has 
been made on this important problem by sub- 



jecting red cell ghosts to a fractionation proce- 
dure in which sodium desoxycholate is used to 
solubilize the membrane proteins. A fraction 
has been obtained which appears to be homog- 
enous by immunochemical criteria and which 
shows a single peak in the analytical ultracen- 
trifuge. The molecular weight is in the neigh- 
borhood of 200,000 to 250,000. Attempts will be 
made to further characterize this fraction. 

Further Studies of the Cholesterol Biosynthetic 
Pathway 

Earlier studies using triparanol as a tool for 
inhibiting cholesterol synthesis suggested that 
A^'-^-cholestedienol was on the normal path- 
way for cholesterol biosynthesis. Radioactive 
A^'^*-cholestedienol was prepared syntheti- 
cally and this has now been shown to be readily 
converted to cholesterol by homogenates of 
rat liver. In addition, it was shown that A^* = 
cholestenol is convertible to cholesterol, al- 
though less readily than the dienol. These 
studies provide further evidence that A^-^*- 
cholestedienol is an intermediate in chol- 
esterol synthesis, and they show further that 
the double bond at the 7 or at the 5 position is 
not an obligatory requirement for the side- 
chain reductase, i.e., the A^*-bond can be re- 
duced even when the steroid nucleus is fully 
saturated. 

Section of Molecular Disease 

Protein Structure and Function 

1. The parathyroid hormone has been suffi- 
ciently purified to begin analysis of its amino 
acid sequence. The immunochemical assay of 
the hormone in plasma has been developed to 
levels of sensitivity and reproducibility suffi- 
cient for study of clinical states of abnormal 
parathyroid function. 

2. Studies of the covalent structure and con- 
formation of ribonuclease and several of its 
smaller derivatives have opened up some 
further methodological approaches to evalua- 
tion of three dimensional conformation of en- 
zymic proteins. 

3. Collaborative studies have extended to 
two years the highly successful treatment of 



116 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the inborn-error of metabolism, cystinuria, by 
the use of penicillamine. 

Plasma Lipids and Lipoproteins 

Several major advances were made during 
the year toward clarifying the nature and 
functional relationships of the plasma lipopro- 
teins and toward understanding clinical abnor- 
malities in their concentrations. 

1. The paper electrophoretic technique of 
Lees and Hatch was set up and thoroughly eval- 
uated. It was concluded this method separates 
lipoproteins into the maximum number of 
groups useful for most clinical purposes at the 
present time. The identify of these four groups 
(alpha, beta, and pre-beta lipoproteins, and 
chylomicrons) was established and cross-corre- 
lated with other means of separating lipopro- 
tein and "particles". For purposes of studying 
pre-beta lipoprotein (endogenous glyceride) 
conditions were determined for reproducible 
augmentation of the concentration of these lipo- 
proteins (carbohydrate-induction) in normal 
subjects and patients. 

An entirely new classification of familial hy- 
perlipoprotenemia was then proposed. It is 
based on the lipoprotein patterns in plasma of 
patients. The conditions include a fasting 
sample and that the patient shall have been on 
a "normal diet". Five different patterns have 
been seen recurrently and correlate with other 
clinical features in such a way as to suggest 
that different genotypes are represented. They 
have been assigned simple designations — Types 
I - V. Use of this systematic approach has al- 
lowed us to segregate what appear to be more 
different diseases among this group of dis- 
orders than has ever been recognized. The 
system has already been adopted by a number 
of laboratories here and abroad. It will serve as 
a frame of reference for future revision and 
expansion. 

2. As pre-beta lipoprotein levels were ob- 
served concomitant with those of alpha and 
beta lipoproteins it became apparent that pre- 
beta lipoprotein (very low density lipopro- 
teins) might consist of beta lipoprotein plus 
alpha lipoprotein plus triglyceride. This was 
confirmed by a number of experimental ap- 
proaches, including decomposition of pre-beta 



lipoproteins and immunochemical identification 
of the products. It was then shown that sub- 
jects having no beta lipoprotein could neither 
be carbohydrate-induced nor make pre-beta lipo- 
protein. Patients deficient in alpha lipopro- 
tein have an increase in plasma glycerides but 
cannot make pre-beta lipoprotein. 

From this and other evidence a tentative 
functional view of plasma lipoproteins has 
been advanced that considers the problem of 
neutral fat transport in terms of three major 
variables, alpha and beta proteins (each having 
a certain complement of cholesterol and phos- 
pholipid) and triglyceride. This represents a 
simplification of lipoproteins to what might be 
called the irreducible minimum of components. 
This provides a structure within which other 
peptides (reported by other laboratories) and 
various subfractions as obtained by density dif- 
ferences, et cetera, can be examined in terms of 
specificity and functional essentiality. These 
views are being summarized in a major review. 

Section on Chemistry 

The Section on Chemistry is pursuing three 
main lines of activity. 7. The structural anal- 
ysis, synthesis and biosynthesis of new com- 
pounds of biological origin. 2. Development of 
methods of analysis for compounds of biologi- 
cal importance. 3. Rendering assistance via our 
organic chemical approach to biological and 
medically oriented groups throughout the In- 
stitutes. Purchase of a new mass spectrometer 
has accentuated the second and third items 
during the recent year as we have attempted to 
educate other groups to its inherent analytical 
utility. 

Specifically, during the past year, individuals 
in this section, either alone or in collaboration 
with others, have: 

1. Synthesized labeled intermediates re- 
lated to phytol for a study of Refsum's 
disease. 

2. Identified acids resulting from the ra- 
diolysis of glycine. 

3. Investigated the analysis of castor oil. 

4. Identified an isomer of elaidic acid. 

5. Proved the structure of a new estro- 
gen, = llj8-estetrol. 



NATIONAL HEART INSTITUTE 



117 



6. Developed a method of analysis of es- 
trogenic ketones in urine. 

7. Confirmed the structure of a new an- 
algesic of the benzomorphinan series. 

8. Assisted in the structure proof of an 
insect sex attractant. 

9. Developed and proved the biosyn- 
thetic pathway of formation of the 
methylene-bis-phloroglucinols. 

10. Revised the structure of a series of al- 
kaloid degradation products. 

11. Assigned structures to two new iso- 
flavones, milletine A and B. 

12. Developed methods for the characteri- 
zation of fern phenols. 

13. Synthesized a structural isomer of the 
alkaloids mesembrine and crinine. 

14. Initiated a mass spectral study of ses- 
quiterpene lactones. 

15. Introduced evidence indicating the 
need for caution in interpreting mass 
spectra via the element map techniqu. 

16. Developed and proved the structure 
of several new alkaloids of Astrocasia 
phyllanthoides. 

17. Developed methods for the future pu- 
spectra via the element map technique, 
rification of components of the kalli- 
krein systems from human urine pan- 
creas and blood. 

18. Proved the location of deuterium in- 
troduction into demosterol. 

In the future, it is assumed that increasing 
emphasis will be placed on biosynthesis and nat- 
ural product structural analysis employing the 
techniques of n.m.r. and mass spectroscopy. 

LABORATORY OF CLINICAL 
BIOCHEMISTRY 

Amine Biogenesis and Metabolism 

Tyrosine hydroxylase has been purified 400 
to 500 fold from beef adrenal medulla. The 
purified enzyme shows a definite requirement 
Fe*+ and reduced tetrahydropteridines which 
is apparently a characteristic of many other 
aromatic hydroxylases. The same enzyme can 
also convert L-phenylalanine to tyrosine and 
the latter to DOPA. The ability of the one en- 
zyme to catalyze two consecutive hydroxylation 



steps offers a means of studying the mecha- 
nism of hydroxylation. 

It appears fairly certain that tyrosine hy- 
droxylase is the rate-limiting step in the over- 
all production of norepinephrine in the sym- 
pathetic nervous system. It has been proven 
that compounds such as a-methyl-tyrosine and 
for 3-iodotyrosine lower tissue norepinephrine 
levels by virtue of inhibiting the initial rate- 
limiting step. In collaborative studies with the 
Experimental Therapeutics Branch, it has been 
shown that administration of a-methyl-tyro- 
sine diminishes the excretion of norepine- 
phrine metabolites in the urine of patients. It 
would appear, therefore, that inhibition of ty- 
rosine hydroxylase will make it possible to 
lower production of norepinephrine in man. 
This has never before been accomplished, by 
chemical means. 

Being the rate-limiting step in the over-all 
process, tyrosine hydroxylase may be suscep- 
tible to other regulatory influences. Some evi- 
dence suggesting changes in norepinephrine 
synthesis in response to sympathetic stimula- 
tion has been obtained. It would also appear 
that the three enzymes involve in norepined- 
phrine formation, tyrosine hydroxylase, dopa- 
carboxylase and dopamine-/3-oxidase, occur in 
the same subcellular fraction and comprise a 
norepinephrine synthesizing (and storage) or- 
ganelle. 

Collagen and Hydroxyproline 

The cell-free system (chick embryo) for con- 
version of proline-C" to collagen hydroxy- 
proline has provided additional information as 
the site of proline hydroxylation. First, by in- 
cubating microsomes with proline-C^**, for 
short periods of time under nitrogen, it is pos- 
sible to incorporate proline-C" and obtain 
very little protein-bound hydroxyproline-C^*. 
Such "proline-prelabeled" microsomes can be 
washed free of diffusible radioactivity and, on 
subsequent incubation in the presence of ascor- 
bic acid, another heat stable cofactor and oxy- 
gen, as much as 10 percent of the protein- 
bound proline is converted to hydroxyproline. 
Furthermore, the "proline-prelabeled" micro- 
somes can be treated with a highly specific bac- 
terial collagenase to yield peptides which con- 



118 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



tain proline-C" and little hydroxyproline-C". 
All these observations are consistent with the 
mechanism previously proposed by this labora- 
tory ; proline is activated and incorporated into 
protein (collagen precursor) and is subse- 
quently hydroxylated in peptide linkage. Evi- 
dence for a hydroxyproline deficient collagen 
precursor has now been obtained from many 
tissues other than the chick embryo. 

In collaboration with Dr. Bernhard Witkop, 
it has been possible to show that the hydroxyla- 
tion of proline involves a selective displacement 
of the hydrogen in the 4-trans position of the 
proline ring by the entering hydroxyl group. 
This information was obtained with syntheti- 
cally prepared cis and trans 4-tritioprolines. It 
was also possible to prepare cis and trans 4- 
fluoroprolines and show that both are incor- 
porated into proteins. Of most interest Avas the 
finding that trans fluoroproline is oxidatively 
defluorinated and converted to collagen. 

The circulating "collagen" previously de- 
scribed by us has been purified about 100 fold. 
Studies in the Experimental Therapeutics 
Branch show that this protein rises in patients 
with certain tumors and may be an important 
diagnostic tool. 

Amino Acid Uptake by Animal Tissues 

It has been possible to obtain several synthet- 
ic amino acids which fluoresce in the visible 
region. These compounds, amino-napthylala- 
nines, are taken up by ascites cells in a manner 
comparable to other amino acids. The studies 
are designed to investigate the physical state of 
the fluorescent amino acid within the cell. If it 
is attached to a "carrier" this should show up 
by changes in several parameters such as quan- 
tum yield, excitation maximum, fluorescence 
maximum and rate of depolarization. 

Biosynthesis of Nitrogen Containing Lipids 

Procedures for assay of palmitoyl-ethanola- 
mide have shown that the compound is present 
in highest concentration in brain and liver. 
When labeled palmitic acid, serine or ethanola- 
mine were administered to animals, the isolat- 
ed palmitoyl ethanolamide was found to be la- 
beled. The enzymatic formation of the fatty 
acid amide has presented an enigma since en- 



ergy sources are not required. However, more 
recent studies suggest that the fatty acids first 
combine with protein in some manner and that 
amines such as ethanolamine, tyramine, phen- 
ethylamine are transferred to the fatty acid by 
displacing a protein. These findings may be of 
great significance with respect to fatty acid 
metabolism and transport. 

RNA Metabolism in Brain 

It was observed that the uptake of labeled 
purines, pyrimidines and their nucleosides into 
brain slices varied with age and with the por- 
tion of the brain utilized. Further studies have 
shown that some nucleosides and purine metab- 
olizing enzymes are asymmetrically distribut- 
ed in the brain and that some of these enzymes 
undergo marked increases following birth and 
during growth. The significance of these 
changes and the factors which regulate them 
are under investigation. 

Proteins and Peptides 

With the reagent, dimethyl amino naptha- 
lene sulfonyl chloride (DANSYL) , it has been 
possible to convert vasoactive peptides to their 
corresponding DANSYL derivatives which are 
highly fluorescent. With the help of this proce- 
dure, it has been possible to isolate and charac- 
terize the major vasoactive peptide from wasp 
venom. The amino acid composition has been 
determined and it has been shown that on 
treatment with trypsin the peptide is cleaved 
to yield a smaller vasoactive peptide which has 
been identifled as glycyl bradykinin. Studies on 
the over-all structure of the mother peptide are 
being continued. It is of interest that the bra- 
dykinin peptide sequence exists in the wasp. 

The presence of j8-aspartyl residues in colla- 
gen has been verified. /3-Aspartyl peptides have 
also been found in fibrin and evidence has been 
accumulated to indicate that j8-aspartyl link- 
ages are increased in the conversion of soluble 
to insoluble fibrin. Evidence has also been ob- 
tained which indicates that the lathyritic 
agent, j8-aminopropionitrile, reacts with /8- 
aspartyl crosslinkages in both collagen and 
fibrin. 



NATIONAL HEART INSTITUTE 



119 



It has been established that the formation of 
the peptide antibiotic actinomycin involves 
mechanisms distinct from the known mecha- 
nisms for protein synthesis. Using D-valine as 
an inhibitor of actinomycin synthesis, evidence 
has been obtained for the accumulation of pre- 
cursors of the phenoxazinone ring. 4-Methyl-3- 
hydroxyanthranilic acid has been detected as an 
intermediate in actinomycin synthesis and ap- 
pears to accumulate during D-valine inhibition. 
It appears that D-valine either prevents the 
synthesis or attachment of the pentapeptide 
chains to the phenoxazinone chromophore. It 
has been shown that the bulk of added D-valine- 
C" which remains in the medium after incu- 
bation occurs as N-succinyl D-valine, the bal- 
ance as D-valine peptides. 

Aromatic Hydroxylation and Metabolism 

The L-phenylalanine hydroxylase from Pseu- 
domonas sp. has been purified to the extent of 
showing an absolute requirement for Fe++ and 
tetrahydropteridines. Bacterial extracts have 
been shown to contain a factor which can re- 
place synthetic tetrahydropteridines as a cofac- 
tor. On further investigation, it was shown 
that the organism can make a hydroxylation 
cofactor from guanosine triphosphate. This 
conversion is being investigated. The bacterial 
cofactor appears not to be exactly the same as 
the cofactor for liver phenylalanine hydroxyl- 
ase. 

Studies were also carried out on some of the 
enzymes involved in the de novo biosynthesis of 
aromatic amino acids in Pseudomonas sp. The 
enzyme, prephenate aromatase, was purified 
and its properties determined. It was also 
shown that addition of phenylalanine to the 
medium not only induces phenylalanine hy- 
droxylase, but also inhibits prephenate aroma- 
tase; added tyrosine stimulates the aromatase. 
All these findings suggest that there exist regu- 
latory mechanisms for turning off aromatic 
amino acid formation in Pseudomonas sp. 
when phenylalanine is present in the medium. 

5-Tritium labeled tryptophan has been 
prepared and is being investigated as a sub- 
strate for tryptophan hydroxylase. If it is suc- 
cessful, it should provide a rapid, simple and 
sensitive procedure for measuring this enzsone. 



Vitamin B12 and Methionine 

Additional evidence has been obtained that 
reduction and alkylation are essential features 
of the cobamide function in methionine bio- 
synthesis. Alkylation of the cobamide has been 
achieved by chemical means; the requirements 
for chemical alkylation are identical to those 
for enzymatic alkylation with Nj-methyl-folate 
in that both require a reducing system and S- 
adenosylmethionine. 

Studies have continued on the control of one- 
carbon metabolism in E. coU. It has been found 
that the metabolism of NsMm-methylene tetra- 
hydrofolate is affected by methionine and pu- 
rines such as guanine. The former represses 
conversion of the methylene folate to the meth- 
yl derivative, the latter represses the conver- 
sion of methylene folate to the methenyl folate. 
These represent mechanisms by which the cell 
regulates methionine and purine biosynthesis. 

Section of Biochemical Genetics 

Although the base composition of UNA co- 
dons and many properties of the genetic code 
were clarified with the use of synthetic polynu- 
cleotides, the sequence of the bases within each 
codon remained unknown. During the last year 
we found a general method of great simplicity 
for determining the base sequence of codons 
and for investigating many aspects of codon 
recognition. The method depends upon the abil- 
ity of trinucleoside diphosphates to direct the 
binding of C"-aminoacyl-sRNA to ribosomes 
prior to peptide bond synthesis. Thus, codon 
recognition was studied with trinucleotide tem- 
plates. Since tri- but not dinucleotides served 
as templates in this system, a triplet code was 
demonstrated directly. 

Since 5'-terminal, 3'-terminal, and internal 
codewords differ in chemical structure, three 
corresponding classes of codewords were pro- 
posed. The recognition of each class in this 
system was studied. The template efficiency of 
trinucleotide codewords was found to be mod- 
ified greatly by terminal phosphate. Triplets 
with 5'-terminal phosphate were more active as 
templates than triplets without terminal phos- 
phate. Triplets with 3'— or 3' (2') -terminal 
phosphate were markedly less active as tern- 



120 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



plates. The modification of RNA and DNA 
codewords, converting sense into missense or 
nonsense codewords, was suggested as a pos- 
sible regulatory mechanism in protein syn- 
thesis. 

Enzymatic methods were devised by M. Bern- 
field and by P. Leder, M. Singer and R. Brim- 
acombe to synthesize trinucleotides of known 
sequence, for two-thirds of the 64 possible tri- 
nucleotides are new compounds, not previously 
synthesized or isolated from RNA digests. 
Thus far we have prepared 45 trinucleotides 
and investigated their template activity for 
C"-aminoacyl-sRNA in this system. Nucleo- 
tide sequences of RNA codons for every amino 
acid have been demonstrated and functions 
have been ascribed to almost every triplet. 

Recent results have permitted several gener- 
alizations to be made concerning the nature of 
the code. (A) Three classes of synonym codon 
sets have been found: 1) synonym codon pairs 
such as XpYpU and XpYpC, 2) synonym pairs 
such as XpYpA and XpYpG and 3) synonym 
codon sets such as XpYpU, XpYpC, XpYpA 
and XpYpG. Recognition of the 3'-terminal 
base clearly is most variable. In one or two 
cases, U = C at the 5'-terminal position. (B) 
In most cases the apparent template activity of 
one member of a synonym codon set differs 
from that of another. (C) Amino acids, which 
are structurally or metabolically related, often 
synthesized in vivo from a common precursor, 
have strikingly similar RNA codons. Such rela- 
tionships would appear to reflect either the 
evolution of the code or direct interactions be- 
tween amino acids and bases in codons. (D) 
Partial recognition of a triplet or alternate rec- 
ognitions of bases possible may suffice during 
protein synthesis. 

The capacity of trinucleotides to direct the 
binding of sRNA to ribosomes and the ease 
with which the process can be assayed provide 
a method for studying the base sequence and 
genetic function of each triplet codon. In addi- 
tion, this method permits the detailed study of 
interactions between codons, sRNA, and ribo- 
somes during the codon recognition process, 
and may provide opportunities to investigate 
mechanisms which control the rate of protein 
synthesis. 



CUNICAL ENDOCRINOLOGY BRANCH 

The activities of the Clinical Endocrinology 
Branch have involved studies on the adrenal 
cortex, studies of renal function with special 
reference to sodium, studies of calcium and 
phosphorus metabolism and miscellaneous 
other studies. 

Adrenal Cortical Steroids 

Patients with hypertension were studied to 
determine the presence or absence of aldoster- 
onism. Aside from the direct measurement of 
excretion of the indirect measurement of secre- 
tion of aldosterone, the production of hypokale- 
mia by means of a high sodium intake 
continues to be the best clinical index for aldo- 
steronism. In each patient with aldosteronism a 
number of variables were evaluated in the at- 
tempt to gain insight into the mechanism of al- 
dosteronism resulting from adrenocortical hy- 
perplasia as opposed to that resulting from 
adrenocortical tumor. Plasma renin was meas- 
ured in all cases and in addition, extensive 
studies of renal hemodynamics with special 
reference to difference between the kidneys, 
and measurements of blood volume and its 
change with sodium intake were performed. 
Each patient with aldosteronism is eventually 
subjected to exploration. Whereas no criteria 
presently available clearly distinguish the two 
types of aldosteronism, the search continues 
for physiologic maneuvers which may suppress 
the aldosteronism of hyperplasia. Suppression 
of aldosterone secretion by the use of 11-hy- 
droxylase inhibitors together with ACTH inhib- 
itors has been carried out for periods up to 30 
days without demonstrable effect on blood 
pressure in patients with aldosteronism of 
either cause. 

Studies on the metabolic pathways for bio- 
synthesis of adrenal cortical steroids in the 
adrenogenital syndrome suggest that the con- 
ventional explanation for the difference be- 
tween salt-losing and non-salt-losing adrenogen- 
ital syndrome (simple difference in "degree" of 
inhibition of 21 -hydroxylase) is inadequate. 
Patients with the non-salt-losing syndrome 
were found to secrete quantities of aldosterone 
far greater than normal and to increase this 
secretion abnormally with physiologic stimuli 



NATIONAL HEART INSTITUTE 



121 



such as sodium deprivation. This has been in- 
terpreted to indicate adequate (and indeed ex- 
cessive) 21-hydroxylation of progesterone in 
such patients, the sole enzymatic defect ap- 
pearing to be a failure of 21-hydroxylation of 
17-hydroxyprogesterone. Presumably, patients 
with salt-losing adrenogenital syndrome have a 
block in 21-hydroxylation of progesterone as 
well. Studies of the control of steroidogenesis 
by the adrenal cortex include a direct compari- 
son in vivo and in vitro of the effect of sodium 
deprivation with that of angiotensin. Unless 
sodium deprivation in fact increases adrenal 
cortical steroid secretion via the hypersecretion 
of renin and of angiotensin, it should be pos- 
sible to distinguish the sites of the effects of 
these two agents in biogenetic pathways for al- 
dosterone. Results suggest that the mecha- 
nisms of actions of the two agents are indeed 
different, as the addition of a precursor "be- 
low" pregnenolone could inhibit aldosterone 
production in the presence of angiotensin 
whereas it could stimulate aldosterone produc- 
tion in the adrenal cortex under the stimulus of 
salt deprivation. According to current con- 
cepts, this strongly suggests that sodium depri- 
vation has an action beyond the site of incorpo- 
ration of a5 pregnenolone into the biogenetic 
pathway for aldosterone. 

In pursuit of the observations that insuffi- 
ciency of carbohydrate-active steroids produces 
marked increases in sensitivity of taste and 
smell and moderate elevations in the rate of 
nerve conduction velocity, studies were insti- 
tuted in cats before and after adrenalectomy. 
The increase of nerve conduction velocity fol- 
lowing adrenalectomy was confirmed. Prelim- 
inary results with extraction of nerve and 
brain tissue before and after incubation sug- 
gest that Cortisol, corticosterone and aldoste- 
rone are contained in nerve tissue and may be 
released from nerve tissue during incubation. 
The steroid content of nerve tissue did not ap- 
pear to be significantly decreased after adre- 
nalectomy despite clear evidence of adrenal 
insufficiency. Since patients with the adreno- 
genital syndrome were found also to exhibit the 
increase in sensitivity for taste and smell char- 
acteristic of patients with adrenal cortical hypo- 
function specifically lacking carbohydrate-ac- 
tive steroids, these thresholds as well as that of 



hearing were measured in such patients before 
and after administration of carbohydrate-ac- 
tive steroids. The patients showed increased 
sensitivity of all thresholds measured; they 
could detect sound frequencies both below and 
above the normal range. Whereas carbohy- 
drate-active steroids returned their taste and 
smell sensitivity to normal (albeit at a rate sig- 
nificantly lower than that for classical Addi- 
son's disease), it had no effect on the hearing 
sensitivity. 

The relationship of hormones to the function 
of nerve cells was further pursued by tissue 
culture procedures in which glial cells and 
nerve cells could be studied separately. Prelim- 
inary studies indicate that carbohydrate-ac- 
tive steroids may increase the cloning efficiency 
of cultured HeLa cells; effect on nerve cells is 
under investigation. In further pursuit of the 
relationship between taste and smell sensitivity 
and hormonal function, detection and recogni- 
tion thresholds for taste and smell were meas- 
ured in a wide variety of clinical syndromes. 
Temporary adrenal insufficiency medicamen- 
tosa produced with metapirone which inhibits 
11-hydroxylation of adrenocortical steroids, 
was associated with immediate increases in 
sensitivity for taste and smell. This appeared 
to be a drug effect as it was not prevented by 
the simultaneous administration of hydrocorti- 
sone. Studies on patients with hypertension 
confirmed previous evidence from this labora- 
tory that taste and smell for sodium and other 
modalities are normal in hypertension and ex- 
hibit normal circadian variability. Study of 
smell sensitivity was pursued in a wide variety 
of patients without known abnormality of 
adrenocortical function. It was found that an- 
osmia and hyposmia do occur in six syndromes 
which can be distinguished from each other. In 
the first there is true anosmia and there may 
be hypogonadism but the disorder is not famil- 
ial. In the second there is marked hyposmia 
which is familial. In the third there is moder- 
ate hyposmia which is familial and there is also 
hypogonadotropic hypogonadism. The fourth 
syndrome is associated with surgical removal 
of primary and secondary centers for taste and 
smell, the fifth with anatomical abnormalities 
of the mid-line including cleft palate and other 
oral defects and the sixth includes the malab- 



122 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



sorption syndrome in which there is hypovita- 
minosis A. In none of these disorders has 
adrenocortical dysfunction been established. 

The gross deficiency in taste sensitivity of 
patients with familial dysautonomia was found 
to depend in part upon absence of circumval- 
late and fungiform papillae in which the taste 
buds are found ; it was further found that par- 
asympathomimetic agents could return taste 
sensitivity for a short period. This indicates 
that some ability to taste is present without 
specific taste buds. 

Studies of Renal Control of Salt and Water 
Metabolism 

We have shown that suppression of adrener- 
gic activity with blocking agents such as guan- 
ethidine promotes sodium loss during sodium 
deprivation and limits the sodium retention re- 
sulting from the use of sodium-retaining ster- 
oids. The results thus suggest a normal role for 
catecholamines in the renal control of sodium 
excretion. This role was further examined in 
patients with various types of disorder involv- 
ing formation of edema and in dogs in which 
pathological retention of sodium and water 
was induced surgically and pharmacologically. 
In addition, the elfects of bradykinin were test- 
ed and compared with those of agents known to 
affect renal sodium excretion. In patients with 
a tendency to edema formation, beta adrener- 
gic blockade with propanolol induced no 
further exacerbation of sodium retention, nor 
did the beta adrenergic blockade appear to de- 
crease the sodium retention resulting from the 
use of sodium-retaining steroids. Alpha adre- 
nergic blockade with dibenzylene on the other 
hand, as well as blockade of both alpha and 
beta receptors with guanethidine decreased the 
sodium retention induced by sodium-retaining 
steroids with two exceptions : In both of the ex- 
ceptions sodium retention was aggravated by 
blockade ; this finding led to further studies of 
cardiac function which was found deficient in 
both patients. The results were taken to indi- 
cate that the dependence of the myocardium on 
catechol activity, unmasked by adrenergic 
blockade, induced sodium retention despite 
blockade of renal alpha receptors as well. 



Dogs were treated with constriction of the 
superior vena cava and given sodium-retaining 
steroids to induce rapid formation of ascites. 
Sodium diuresis was induced in such dogs with 
ganglionic blockade produced with pentolinium 
tartrate ; in a number of instances, there was a 
decrease of mean arterial pressure and a de- 
crease in the glomerular filtration rate, find- 
ings which indicate clearly a role for the 
adrenergic nervous system in tubular reabsorp- 
tion of sodium. Bradykinin could also increase 
sodium excretion despite decreases in glomeru- 
lar filtration rate in normal subjects; the so- 
dium loss could be prevented by adrenergic 
blockade. The results suggest a complex role 
for bradykinin which may involve release of 
angiotensin or of renin. 

The relationship of hormonal and nervous 
activity to renal control of sodium excretion 
could not be clarified without more intimate 
knowledge of renal circulation and studies 
were instituted to measure cortical and medul- 
lary flow separately. With the use of Xenon^^^ 
injected into the renal artery, exponential de- 
cay curves were derived which, upon compart- 
mental analysis, could provide indices of blood 
flow in various parts of the renal circulation. 
The application of this method to the variables 
under study is in progress. 

The special natriuresis which accompanies 
fasting was further studied by metabolic bal- 
ance in human subjects. The natriuresis could 
not be attributed to Ketone body formation 
alone, although it appeared likely that these 
studies contributed in supplying anions requir- 
ing excretion. Results suggest that the natriu- 
resis results in part from a failure of hyperse- 
cretion of aldosterone which normally results 
from a decrease in sodium intake. It appears 
further that potassium depletion may contrib- 
ute to this failure of aldosterone secretion. 
Finally, it appears that carbohydrate alone can 
restore the ability to secrete aldosterone nor- 
mally. The mechanism for this function of car- 
bohydrate is under study. 

Studies of the salt and water excretion in the 
syndromes of inappropriate secretion of anti- 
diuretic hormone were continued in a 5-year 
old child with a severe gunshot wound in the 
region of the hypothalamus. Inappropriate se- 
cretion of antidiuretic hormone was document- 



NATIONAL HEART INSTITUTE 



123 



ed and shown to disappear with the passage of 
time. As the patient had developed a marked 
resistance to the stimulus of metapirone to 
secretion of ACTH, it was suggested that a 
deficiency of Cortisol may play a part in the syn- 
drome. If the inappropriate secretion of anti- 
diuretic hormone results from the hypothalamic 
lesion, it is possible that other patients with 
the syndrome may demonstrate an inability to 
respond to metapirone. 

In pursuit of the relationship between so- 
dium excretion and adrenergic activity, exten- 
sive preliminary studies of epinephrine and nor- 
epinephrine excretion have been carried out in 
patients with a wide variety of diseases. The 
normal range for excretion of free and bound 
epinephrine and norepinephrine has been es- 
tablished ; data for all age groups can be fitted 
to a single scale when "corrected" for surface 
area. Normal excretion of free and bound epi- 
nephrine and norepinephrine was shown to per- 
sist under the influence of ACTH and in pa- 
tients with congenital adrenal hyperplasia, 
panhypopituitarism, acromegaly, hypoparathy- 
roidism, hyperthyroidism, pseudohypoparathy- 
roidism and idiopathic edema; normal values 
for free and bound epinephrine and norepi- 
nephrine were found in one patient with 
sub-total and one with total adrenalectomy, in- 
dicating a non-adrenal source for urinary epi- 
nephrine. Bound epinephrine was found to be 
very low in urine of patients with familial dys- 
autonomia. Bound norepinephrine was the 
single consistent abnormality of the epineph- 
rine-norepinephrine excretion of the patients 
with pheochromocytoma. The relationship of 
catecholamine excretion to the sate of sodium 
balance is under investigation. 

Studies of Calcium and Phosphorus Metabolism 

A large number of studies of the distribution 
of calcium^^ have been carried out with concom- 
itant observations of the specific activity of 
blood, urine and stools in an attempt to con- 
struct a model which will explain the events in 
metabolic bone diseases and in patients with 
various diseases with and without treatment 
such as sarcoidosis with and without vitamin 
D, osteoporosis with and without estrogen, and 
in normal subjects to measure the effect of cal- 



cium loading, of phosphate deprivation, and of 
growth hormone. Studies of conventional dy- 
namics show marked acceleration of bone turn- 
over in patients with sarcoidosis, the change 
having the same characteristics as that induced 
by vitamin D in normal subjects. This finding 
suggests that the bones, like the gastrointesti- 
nal tract in sarcoidosis, may suffer hypersensi- 
tivity to vitamin D. Calcium infusion markedly 
elevated calcium turnover in normal subjects, a 
finding that suggests a simple mass action at 
bone surfaces and one which indicates the need 
for considerable caution in the interpretation 
of conventional dynamic data. Simple hyper- 
calcemia may introduce spurious variation. 

Phosphate deprivation was found to alter 
bone dynamics profoundly in normal subjects, 
inducing extremely rapid bone turnover, in- 
creases in serum calcium and marked increases 
in urinary calcium. Indeed, it was found that 
in hypoparathyroidism phosphate deprivation 
alone could restore serum calcium to normal 
over long periods and increase urinary calcium 
to super-normal quantities over the same pe- 
riod. The dynamic data indicate further that 
phosphate deprivation induced by Maalox-am- 
phogel which lowers urinary phosphorus to 
near-zero values increases the absorption of 
calcium in the gastrointestinal tract. At the 
same time, it increases skeletal resorption of 
calcium. 

The use of urinary hydroxyproline as an in- 
dex of collagen turnover has proved of great 
value in assessing metabolic bone disease. An 
unexpected finding developed with the experi- 
mental elevation and depression of serum cal- 
cium in hyperparathyroidism. It had been ex- 
pected that this would not alter hydroxyproline 
excretion if parathyroid hormone secretion 
was not changed. Consistently, the opposite 
was true. A survey of patients to define the in- 
trinsic gastrointestinal ability to absorb cal- 
cium was continued. The majority of normal 
subjects show little change in calcium absorp- 
tion and in urinary calcium as dietary calcium 
is increased twenty-fold. Rare normal subjects 
and a number of subjects with a tendency to 
stone formation show linear increase in uri- 
nary calcium with increases in dietary calcium. 
It appears likely that the two groups are genet- 
ically determined. 



124 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Studies designed to clarify that hypophos- 
phatemia of rickets and the effect of vitamin D 
in restoring it have been continued with clini- 
cal studies in hypophosphatemic rickets and 
with experimentally-produced rickets in pup- 
pies. In the latter, it was clearly shown that the 
hypophosphatemia of rickets depends on sec- 
ondary stimulation of parathyroid activity 
which results from the hypocalcemia produced 
by deficiency of vitamin D. In the rachitic pup- 
pies in which the abnormalities of phosphorus 
metabolism (decrease of serum phosphate and 
increase of phosphate clearance) quickly 
returned towards normal after parathyroidec- 
tomy, exogenous parathyroid extract has its 
usual effects, a finding which negates early 
reports that parathyroid hormone requires vi- 
tamin D for its effects. Hydroxyproline excre- 
tion was found to be elevated in the rachitic 
puppy, a finding in support of the view that 
urinary hydroxyproline results in part at least 
from release of soluble pre-collagen associated 
with bone formation and not exclusively from 
the release of collagen as bone is destroyed. 
There is no evidence of bone destruction in the 
rachitic bones of these animals. 

Miscellaneous Studies 

The development of atherosclerosis was stud- 
ied by measurement of the rate of acumula- 
tion of I"^ labeled lipoprotein in the aorta of 
rabbits before and after the production of ath- 
erosclerosis and hyperlipidemia. Development 
of atherosclerosis was associated with marked 
increase in accumulation of lipoprotein sug- 
gesting that such increase of permeability may 
play a part in the development of atheroscler- 
osis. 

Numerous studies of circadian rhythm with 
especial reference to variables associated with 
the adrenal cortical function have been carried 
out in normal subjects and some in patients sus- 
pected of diseases of desynchronization such 
as those with periodic disorders. Transverse 
maps of adrenal cortical variables were carried 
out in Australia to verify the essential similar- 
ity of the external and internal timing of 
adrenal events at 145° east longitude to those 
in the United States. In a patient with periodic 
fever and hypertension, extensive studies of 



hemodynamic and adrenal events suggested 
that different periods may appear for grip 
strength, blood pressure, pulse and respiration 
as opposed to time estimation. Such a differ- 
ence could be a first indication of a disease of 
desynchronization. 

In pursuit of other indices for the measure- 
ment of periodic hyperpyrexia, the branch has 
developed and tested extensively a new method 
for the measurement of serum etiocholanolone, 
the first of the five-beta steroids implicated in 
the disorder of periodic hyperpyrexia. Elevated 
values were indeed found in a patient with car- 
cinoma of the rectum but normal values have 
been found in a number of patients with period- 
ic fever; thus far, no confirmation of the syn- 
drome of high fever resulting from excess of 
plasma etiocholanolone has been obtained. 

Effect of penicillamine in cystinuria was 
studied by clinical and biochemical means. It 
was shown that penicillamine decreased effec- 
tively and consistently the urinary cystine in 
patients with cystinuria; surprisingly, it pro- 
duced also a decrease in total half -cystine, that 
bound to penicillamine being much less than the 
amounts originally present in the urine. In ex- 
tensive clinical trials of the effect of penicilla- 
mine it was found possible to control stone for- 
mation effectively and to institute the dissolu- 
tion in vivo of stones in four subjects. 

Studies of the abnormalities of renal func- 
tion in cystinosis were continued. The effect of 
penicillamine in decreasing the basic storage of 
cystine is under study. It has been found that 
patients with cystinosis show also a poor re- 
sponse to metapirone, a finding suggesting ad- 
ditional disorder of pituitary function, possibly 
related to the growth defect characteristic of 
this syndrome. 

A disorder in infants characterized by hy- 
percalcemia but differing from the classical 
syndrome of "idiopathic hypercalcemia of in- 
fants" in the association of hyperlipemia and 
hypercalcemia has been studied in two sub- 
jects. Hyperlipemia was shown to be dependent 
on dietary carbohydrate. It was found that hy- 
percalcemia was not directly dependent upon 
the elevation of blood lipids. The elevation of 
blood lipids may reflect the abnormality in vi- 
tamin D metabolism which in turn may be re- 
lated to the hypercalcemia. 



NATIONAL HEART INSTITUTE 



125 



The finding that the serum and dialysis fluid 
of patients with uremia may contain "toxic" 
substances was pursued. A previous observa- 
tion that HeLa cells are killed by the eluates of 
such fluid after passage through Sephadex was 
confirmed. The toxic material appeared in two 
separate peaks during the elution. The uptake 
of glucose by normal red cells was effectively 
blocked by the peaks which have a toxic effect 
on HeLa cells and could kill normal mice. Con- 
tinued studies are designed to identify the tox- 
ic substances. An unexpected finding developed 
during prolonged peritoneal dialysis of a pa- 
tient with vitamin D intoxication in the dem- 
onstration that over a million units of vita- 
min D were removed directly by dialysis. 

EXPERIMENTAL THERAPEUTICS BRANCH 

A variety of investigations chiefly of imme- 
diate or at least potential clinical significance 
have been continued. Findings obtained will be 
considered under the following headings: 1) 
Biochemistry and Pharmacology of Vasoactive 
Amines, 2) Studies on the Kinin Peptides, 3) 
Chemistry of Selected Proteins and 4) Miscel- 
laneous. 

Biochemistry and Pharmacology of Vasoactive 
Amines 

It was shown previously that single doses of 
two inhibitors of histidine decarboxylase (s), 
4-bromo-3-hydroxybenzyloxyamine (NSD 1055) 
and the a-hydrazino analogue of histidine 
(MK 785), produced marked decreases in 
urinary and tissue histamine levels in female 
rats, after intraperitoneal injection. In subse- 
quent studies it was found that only slight de- 
creases in histamine levels in guinea pig tissues 
and urine occurred even after repeated admin- 
istration of these compounds. Further, no 
changes were observed in rats following oral 
dosing. Since leaving the department July 1, 
1964, Dr. R. J. Levine has shown an inhibitory 
effect of these agents on gastric secretion in 
the rat. We have observed no changes in gas- 
tric secretion or urinary histamine in human 
subjects, however, following short term admin- 
istration of NSD 1055 orally. Additional stud- 
ies are planned with both agents in patients. 

The first step in the biosynthesis of serotonin 



is the hydroxylation of tryptophan to form 
5-hydroxytryptophan. A tryptophan hydroxy- 
lase has been partially purified from neoplastic 
mouse mast cells and characterized. The en- 
zyme is a typical aromatic ring hydroxylase 
and has an absolute requirement for oxygen, 
with a tetrahydropteridine as a specific reduc- 
ing source. It is unusual in showing a nearly 
complete and specific dependence on ferrous 
iron. Although phenylalanine is also a sub- 
strate, the enzyme can be distinguished from 
the phenylalanine hydroxylase of rat liver. It 
has the lowest Km for tryptophan (4xlO-^M) 
of any tryptophan hydroxylating system stud- 
ied to date. Current emphasis is on discovery 
of inhibitors of the enzyme, as a possible ap- 
proach to selective blockade of serotonin bio- 
synthesis in vivo. 

The hydroxylation of tyrosine to dopa has 
been shown previously to be the rate-limiting 
step in the biosynthesis of norepinephrine. Sev- 
eral compounds found by scientists in LCB to 
block tyrosine hydroxylase in vitro have now 
been studied in animals, and preliminary ob- 
servations with two of the compounds have 
been made in man. The first and most potent 
compound studied was a-methyl-p-tyrosine 
(aMPT). Repeated dosage of guinea pigs pro- 
duces almost 100% depletion of tissue norepi- 
nephrine without affecting serotonin levels. 
Numerous experiments indicate the mechanism 
of depletion to be by blockade of synthesis 
though observations with radioactive aMPT in- 
dicate that there is some conversion to a-meth- 
yl-catecholamines, raising the possibility of ad- 
ditional types of action of the compound during 
its prolonged administration. Three other tyro- 
sine hydroxylase inhibitors, 3-iodo-tyrosine, 
3-iodo-a-methyl-tyrosine and a-methyl-phen- 
ylalanine, have also been found to reduce 
endogenous levels of catecholamines but not as 
effectively as with a-MPT. The pharmacologic 
effects observed with a-MPT and to a lesser ex- 
tent with the other inhibitors in cats, rats and 
guinea pigs include : reduced motor activity, se- 
dation, slight meiosis, reduced tyramine pres- 
sor responses and reduced responses of the nic- 
titating membrane (cat) to nerve stimulation. 
Interestingly, no lowering of blood pressure 
has been observed. In a single patient with 
pheochromocytoma, e-iodo-tyrosine in maxi- 



126 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



mum dosage of 2.0 gm/24 hrs. was without 
detectable chemical or pharmacologic effect. 
Alpha-MPT has been given in maximum dos- 
age of 1.2 gm/24 hr. to several patients with 
essential hypertension and also three patients 
with pheochromocytoma ; effects observed in- 
clude marked drowsiness which tends to wave 
with continued treatment, persistent tranquili- 
zation in some patients, significant decrease in 
urinary excretion of vanilmandelic acid 
(VMA) and lowering of blood pressure in the 
patients with pheochromocytoma but not defi- 
nitely in those with essential hypertension. A 
broad clinical investigation of tyrosine hydrox- 
ylase inhibitors in man is contemplated. 

In an attempt to study the turnover rate of 
endogenous norepinephrine in man, H^-dopa 
has been administered to two subjects and the 
specific activity of urinary dopamine and nore- 
pinephrine studied as a function of time. While 
both amines were highly labeled, the specific 
activity of urinary dopamine failed to decrease 
below that of norepinephrine, negating calcula- 
tions of turnover rate for the latter amine. The 
feasibility of calculations of dopamine turnover 
rate are under investigation and the use of 
highly labeled tyrosine is planned. 

Our tyramine pressor test for pheochromo- 
cytoma is now undergoing broad clinical test- 
ing under commercial sponsorship. In personal 
studies on about 200 patients with essential hy- 
pertension, "negative" tests have been ob- 
served. The same was true in five patients with 
renovascular hypertension. Positive tests, i.e., 
20 mm. Hg. systolic blood pressure rise follow- 
ing 1.0 mg (or less) of tyramine intravenously, 
have now been obtained in 12 patients with 
pheochromocytoma. No definite false negative 
tests have occurred. Thus, our initial favorable 
impressions have been sustained for an addi- 
tional year. 

We have been prompted to study in more 
detail the role of diet and intestinal bacteria on 
the excretion of urinary amines. A report in 
the literature implies that elevated levels of 
urinary tryptamine and tyramine during treat- 
ment with a monoamine oxidase (MAO) inhib- 
itor may not reflect changes in the metabolism 
of amines produced in tissues. By use of glu- 
cose diets and oral antibiotics it can be shown 
that the elevated amounts of tyramine and 



tryptamine in urine during MAO inhibition 
are primarily of tissue rather than intestinal 
(luminal) origin. 

Kinin Peptides 

Previous studies have strongly implicated 
the kallikrein system in the pathogenesis of 
flushing in carcinoid patients. While amounts 
of bradykinin capable of producing a flush can 
be measured in hepatic vein blood of such pa- 
tients using routine bioassay techniques, no 
current method is sufficiently sensitive and spe- 
cific to measure the low levels of bradykinin in 
peptides are adsorbed on a CM-Sephadex col- 
been exploring the preparation of fluorescent 
derivatives and separations by thin layer chro- 
matography as the basis for chemical methods 
of measuring vasoactive peptides. Basic 
peptides are adsorbe don a CM-Sephadex col- 
umn, eluted and fluorescent derivatives are pro- 
duced which are then developed on thin layer 
chromatography and quantified in a fluoro- 
meter. It is thought that this chemical approach 
may yield sensitive and specific means of assay 
of bradykinin, as well as other basic peptides 
which may be found in biological materials. 
Further exploration of alterations of kinin pep- 
tides in health and disease is being deferred 
pending the development of improved methodol- 
ogy. 

Chemistry of Selected Proteins 

Measurement of the excretion of hydroxy- 
proline peptides as an index of endogenous col- 
lagen metabolism has been continued in a 
number of disease states. In confirmation of 
previous work, elevations have been observed 
in thyrotoxicosis, Paget's disease, and in acro- 
megaly wherein diagnostic usefulness is appar- 
ent. Levels of a hydroxyproline-containing 
protein in plasma, which we have termed 
"hypro-protein", have been determined in 101 
normal subjects and in 146 patients with a va- 
riety of diseases. Normal levels range from 5 to 
10 jag/ml (expressed as hydroxyproline) with 
little age variation. Elevated levels up to 30 
ju,g/ml occur in a variety of clinical disorders, 
the most consistent and highest elevations be- 
ing in Hodgkin's disease, Sjogren's syndrome, 
and in febrile-inflammatory states. Practical 



NATIONAL HEART INSTITUTE 



127 



use has been made of plasma hypro-protein as- 
says in evaluating the "activity" of Hodgkin's 
disease, the levels returning to normal when 
patients are in remission. Studies on artificial 
fever (etiocholanolone and endotoxin) indicate 
that fever per se is not accompanied by eleva- 
tions in hypro-protein. Studies are in progress 
to purify this protein and establish whether it 
is identical with known macromolecular pre- 
cursors of the collagen fibril. 

Preparatory to the investigation of collagen 
pools in man, the conversion of radioactive pro- 
line to peptide-bound hydroxyproline in tissues 
and urine has been studied in animals. Techni- 
cal difficulties were encountered which we be- 
lieve put into question the validity of many 
reported studies. Modifications in methodology 
are being perfected to assure that only radioac- 
tivity present in hydroxyproline will be meas- 
ured. In another pi"e-clinical study, experi- 
mental lathyrism produced by injection of j8- 
amino-propionitrile (BAPN) has been studied 
as a model of collagen dysfunction. Chemical 
changes include an increase in a-chain subunits 
in skin collagen and elevated excretion of uri- 
naiy hydroxyproline. A protocol for pilot stud- 
ies using BAPN in humans afflicted with 
diseases characterized by excessive collagen 
formation is being explored. 

The ferredoxins are small, non-heme, iron- 
containing proteins which serve as electron 
carriers in low redox potential reactions in 
certain bacteria. The chemical characteristics 
of several clostridial ferredoxins have been ex- 
amined. Ferredoxin from Clostridium pasteu- 
rianum has been examined in most detail ; it is 
composed of a single polypeptide chain of 55 
amino acids to which are bonded 7 molecules of 
iron and 7 molecules of inorganic sulfide. The 
amino acid sequence has been largely deter- 
mined. Experiments in progress indicate that 
ferredoxin is a two-electron rather than a one- 
electron carrier as has been supposed. During 
the studies on ferredoxin, a red protein was 
crystallized from extracts of C. pasteurianum. 
Its electron carrying properties and molecular 
weight were found to be similar to those of 
ferredoxin though it has a lower content of 
iron, lacks inorganic sulfide, and has a some- 
what different amino acid composition. It is of 
great interest to us that non-heme, iron-con- 



taining peptides have recently been described 
in mammalian systems ; explorations in this di- 
rection are in progress. 

Miscellaneous 

1. Evidence has been obtained that the 
mechanism of catecholamine depletion 
produced in animal tissues by metar- 
aminol is not that of stoichiometric 
(mole for mole) displacement as some 
have claimed. 

2. SU 5184, an analogue of reserpine 
which produces selective depletion of 
catecholamines in the tissues of ani- 
mals, has been administered to hyper- 
tensive patients in oral doses as high 
as 96 mg/day. No effects were ob- 
served and studies have been discontin- 
ued. 

3. Effective chronic medical therapy for 
pheochromocytoma using phenoxyben- 
zamine has now been continued for 
periods of 2-3 years in three patients. 

4. A syndrome simulating pheochromocy- 
toma has been recognized in patients 
with angina pectoris. It is postulated 
that pressor crises are due to reflexes 
triggered by reductions in coronary 
flow. 

5. A variety of sympatholytic agents have 
been found to be useful in treatment of 
angina pectoris when drug dosages are 
geared to reduction of cardiovascular 
responses to exercise. 

CARDIOLOGY BRANCH 

Introduction 

The ultimate goal of the research efforts of 
the Cardiology Branch, NHI, is to obtain a 
detailed appreciation of the pathologic physiol- 
ogy which characterizes various forms of clin- 
ical heart disease. It is believed that this infor- 
mation will allow the development of more 
effective approaches to treatment. It is becom- 
ing evident that in order to achieve this objec- 
tive not only are careful clinical and hemody- 
namic studies of patients with various cardiac 
abnormalities necessary, but a substantially 



128 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



greater understanding of the normal myocar- 
dial contractile processes is required. Unless 
the mechanical and energetic processes in- 
volved in normal cardiac contraction can be 
described accurately and in detail, any analysis 
of the diseased heart muscle will of necessity 
be quite superficial. In view of this considera- 
tion, attention is being directed, in the research 
program of the Branch, to normal and abnor- 
mal hearts ; both man and a variety of experi- 
mental animals are being studied, and work is 
carried out on intact hearts as well as on isolat- 
ed portions of heart muscle, as the experimen- 
tal situation demands. It is hoped that this non- 
structured, flexible approach to the study of 
the circulation will ultimately provide addition- 
al information concerning the abnormalities of 
function which occur in clinical heart disease 
and will thereby permit a rational approach to 
treatment. 

Determinants of Myocardial Energy Exchange 

A study has been initiated in order to define 
the O2 requirements of the myocardial mem- 
brane activity responsible for de- and repolari- 
zation. "Electromechanical dissociation" is 
produced in isolated canine hearts by perfusing 
the hearts with blood which has been rendered 
Ca-free. In these hearts the electrical response 
is not associated with any contractile activity. 
Increases in the frequency of depolarization 
have thus far been accompanied by small but 
consistent increases of myocardial O2 uptake, 
averaging 0.4 fxl O2 per depolarization per 100 
grams of heart. These increases are considered 
to represent the maximum amount of O2 that 
may be required for a depolarization and repo- 
larization and approximate only 1% of the O2 
consumed by the normally contracting heart. 

It has been widely held that tension and the 
time it is maintained, the "tension-time index", 
is the major, if not the sole determinant of 
myocardial O2 consumption. However, in view 
of studies on skeletal muscle by A. V. Hill in 
which it has been shown that the rate with 
which energy is liberated by a muscle is pro- 
portional to the maximum velocity of muscle 
shortening, the possibility was considered that 
the velocity of myocardial shortening might be 
an important determinant of myocardial O2 



consumption. In order to explore this possibil- 
ity, the Oo consumption of the dog heart was 
measured under circumstances in which the 
stroke volume, aortic pressure, and heart rate 
could be held constant while the velocity of 
myocardial contraction was augmented by 
distinctly different inotropic interventions; 
norepinephrine, calcium, sustained postex- 
trasystolic potentiation, or a combination 
of these influences. It was found that at a 
constant level of ventricular work O2 consump- 
tion is correlated with the velocity of ventricu- 
lar contraction. This was true regardless of the 
specific inotropic influence by which the veloc- 
ity was augmented. In addition, this augmen- 
tation occurred consistently despite concomi- 
tant decrements in the "tension-time" index. It 
has been concluded that in addition to factors 
related to force or tension, intrinsic velocity of 
the myocardium is an important factor in de- 
termining myocardial O2 consumption. Fur- 
thermore, it now appears likely that the so- 
called O2 wasting eflfect exerted by norepine- 
phrine on myocardial metabolism may be ex- 
plained largely by an increased velocity of con- 
traction. 

Other evidence has also been obtained that 
the increase of myocardial O2 uptake produced 
by catecholamines is due primarily to the aug- 
mentation of hemodynamic activity which the 
amines induce. Using isolated canine hearts, 
the response of O2 uptake to graded doses of 
isoproterenol, norepinephrine, and epinephrine 
was determined before and after the induction 
of arrest with potassium. Increases of O2 up- 
take did occur in the arrested state, but were 
only a small fraction (5 to 20%) of the in- 
creases produced by the same doses of amines 
when the hearts were beating. From these ob- 
servations it appears that although large doses 
of catecholamines can stimulate oxidative 
metabolism directly, the increases of O2 uptake 
which catecholamines produce in a beating 
heart are due primarily to the augmentation of 
myocardial contractile activity which the 
amines induce. 

Previous observations in experimental heart 
failure in animals have indicated that there 
may be a bioenergetic defect in the myocar- 
dium involving an impairment of oxidative 
phosphorylation. This important problem was 



NATIONAL HEAKT INSTITUTE 



129 



studied in myocardial tissue removed from pa- 
tients with heart failure at the time of cardiac 
operations. Mitochondria isolated from left 
ventricular papillary muscles were found to be 
functionally and anatomically normal. Myocar- 
dial creatine phosphate was also found to be 
within the normal range in these patients. It is 
concluded that the formation of chemical en- 
ergy is not impaired in the failing heart, and it 
is suggested that the biochemical abnormality 
responsible for defective myocardial function 
involves utilization of energy in the contractile 
process. 

Mechanics of Myocardial Contraction 

Experimental. Continued effort has been 
directed towards defining the ultrastructural 
basis of contraction in heart muscle and to- 
wards understanding the structural determi- 
nants of contraction in the intact heart. Earlier 
observations on the sarcomere length-tension 
relation established for the papillary muscle 
have been extended to the intact heart. Intact 
left ventricles of both cats and dogs were fixed 
for electronmicroscopic analysis at known vol- 
umes and pressures, and the length of the sar- 
comeres subsequently determined. These stud- 
ies have shown that sarcomere length is a 
function of ventricular pressure, and is inde- 
pendent of absolute ventricular volume. The 
sarcomere length-tension curve is thus a gener- 
alized phenomenon and serves to explain the 
ubiquitous nature of the Frank-Starling rela- 
tion in mammals. At intraventricular pressures 
approximating the upper limits of normal in 
the functioning heart, sarcomere length corre- 
sponds to that seen at the apex of the length- 
tension curve. Therefore, the limit to ventricu- 
lar performance seen with increasing initial 
ventricular volume is determined by the struc- 
ture of the sarcomere. 

A dissociation between thick and thin myofil- 
aments in the sarcomere has also been shown 
to occur in overdistended ventricles. This obser- 
vation, along with evidence that slippage of 
myocardial cells occurs in the overdistended 
ventricle, helps to explain the mechanical de- 
compensation of ventricular performance 
which occurs as a consequence of cardiac dila- 
tation. 



This investigation has recently been ex- 
tended to the living heart. A technique has 
been developed which permits nearly instan- 
taneous arrest and fixation of the left ventricle 
of the dog during any phase of the cardiac 
cycle. The left coronary artery is cannulated 
and attached through a sidearm to a power- 
syringe which suddenly injects glutaraldehyde. 
Subsequent electronmicroscopic studies have 
shown that the length of sarcomeres, arrested 
during diastole are 2.0 to 2.2 microns, while 
those obtained from hearts arrested in systole 
measure 1.6 to 1.8 microns. These measure- 
ments have allowed direct comparisons with 
values obtained in the passively distended ven- 
tricle, as well as with the sarcomere lengths in 
isolated papillary muscle. It is anticipated that 
ultimately the length-tension relationships of 
the sarcomere of the left ventricle will be 
known throughout the entire cardiac cycle and 
under a number of experimental conditions. 

The active state of muscle has been defined 
as a mechanical measure of those processes in 
the contractile elements which generate force 
and shortening. In a study of the course of ac- 
tive state in the cat papillary muscle it was 
found that: 1) In contrast to skeletal muscle, 
the onset of maximum active state in heart 
muscle is delayed, developing 100-150 msec, 
after the first evidence of active state ; 2) maxi- 
mum active state is maintained for about 100 
msec, and does not decline until just prior to 
the development of maximum active tension; 
and 3) inotropic interventions which alter the 
contractile state of the muscle such as norepi- 
nephrine or changing frequency of contraction, 
accelerate the onset, increase the intensity, and 
hasten the decline of the active state. The de- 
layed onset of active state is consistent with 
the view that following electrical stimulation, 
an activator is released from the sarcoplasmic 
reticulum and requires a given time for diffu- 
sion to the contractile sites. 

The inverse relation between force and veloc- 
ity of shortening comprises one of the most 
fundamental properties of the contractile sys- 
tem of muscle. However, it has not been deter- 
mined whether the force-velocity relation per- 
tains during the course of contraction while 
changes in muscle length occur. Using the cat 
papillary muscle, and following the instanta- 



130 



ANNUAL REVIEW OF INTEAMURAL RESEARCH 



1 



neous velocity of muscle shortening during af- 
terloaded isotonic contractions, evidence has 
been obtained to support the view that the ve- 
locity of shortening during contraction depends 
on instantaneous muscle length during the 
course of the contraction and is essentially in- 
dependent of the muscle length at which the 
contraction began. The extension of these re- 
sults has permitted the plotting of the force-ve- 
locity-length relations which serve to define a 
given contractile state of the muscle and allow 
for a more generalized analysis of contraction 
in heart muscle than is afforded by the relation 
between initial muscle length and force and ve- 
locity. 

It is now generally agreed that the myocar- 
dium may be considered in terms of an active 
contractile element arranged in series with a 
passive elastic component. An investigation 
was undertaken to define the characteristics 
of the series elastic element of heart muscle 
and explore its role in the generation of myo- 
cardial force. The stiffness of the series elastic 
was shown to be a linear function of the devel- 
oped force fo contraction. The load-extension 
curve of the series elastic was thus exponential 
in form, the series elastic being stretched an 
amount equivalent to 8 to 10% of initial muscle 
length during the development of maximum 
isometric force. Evidence was also obtained to 
indicate that the series elastic component is not 
changed by interventions which greatly alter 
the activity of the contractile elements such as 
changing the frequency of contraction or add- 
ing strophanthin. 

Until recently, the performance of the heart 
was most commonly described by determining 
relations between the filling pressure of the 
ventricle and its stroke volume or stroke work, 
and by defining alterations in the contractile 
state in relation to a family of ventricular 
function curves. However, since it has been 
shown that the behavior of isolated papillary 
muscle, like that of skeletal muscle, can be char- 
acterized effectively in terms of its force-veloc- 
ity-length relation, an investigation was un- 
dertaken to characterize the performance of 
the intact canine ventricle in terms of its force- 
velocity-length relation. Previous diflaculties in 
constructing force-velocity curves were over- 
come by examining only the first beat after a 



sudden alteration in aortic pressure, the 
change being imposed during diastole. Force- 
velocity curves were constructed by calculating 
the velocity of fiber shortening and wall ten- 
sion at a given systolic volume. It was shovm 
that the force-velocity curves are significantly 
shifted upwards and to the right by positive 
inotropic influences and in the opposite direc- 
tion by acute heart failure. It is suggested that 
these curves may provide a more unified defini- 
tion of the contractile state than heretofore 
possible. 

In order to study with greater precision the 
contribution of atrial contraction to ventricu- 
lar filling, a highly sensitive, adjustable strain 
gauge arch was sewn to both atria of dogs, and 
atrial length-tension curves were determined. 
It was found that the length-tension character- 
istics of the atria are similar to those of the 
ventricle, and that influences Avhich exert a pos- 
itive inotropic action on the ventricles, i.e., 
sympathetic stimulation, directly or reflexly in- 
duced, and the administration of calcium, iso- 
proterenol, and acetyl strophanthidin, also in- 
crease atrial force. Since atrial contraction 
contributes significantly to ventricular filling, 
an increased force of atrial contraction may 
represent another mechanism utilized by the 
heart to meet increased demands imposed upon 
it and these studies have clarified some of the 
determinants of the force of atrial contraction. 

It is not known whether an alteration in 
myocardial distensibility occurs with the devel- 
opment of hypertrophy. It has been the general 
impression that the hypertrophied ventricle 
is less distensible than normal, either because 
of an increase in the total muscle mass or a 
decreased compliance of individual fibers. This 
is an important question since hypertrophy is 
such a fundamental response of the diseased 
myocardium. Myocardial hypertrophy was pro- 
duced in rats by subdiaphragmatic suprarenal 
aortic constriction. It was found that hyper- 
trophied myocardium is similar to normal myo- 
cardium in its resting length-tension relation- 
ship when suitable corrections are made for 
differences in muscle mass and length. From 
these data it would appear that hypertrophy 
alters myocardial compliance simply by in- 
creasing muscle mass, rather than producing a 
qualitative change in the myocardium. 



NATIONAL HEART INSTITUTE 



131 



The effects on the myocardium of paired 
electrical stimulation were studied in detail. In 
paired electrical stimulation of the heart, two 
stimuli are repetitively delivered in such a 
manner that the second stimulus of each pair is 
introduced immediately after the termination 
of the absolute refractory period. If the second 
stimulus of each pair is sufficiently early, it 
evokes a propagated ventricular depolarization, 
but a contraction so weak that it is barely evi- 
dent. Paired electrical stimuli were applied to 
the ventricle of dogs with sinus tachycardia. In 
each experiment the ventricle could be cap- 
tured by the electrical pacemaker, and the 
number of effective contractions was reduced 
from an average of 152 to 109 per minute. 
When paired electrical stimulation was discon- 
tinued, the underlying sinus tachycardia re- 
curred. Similar alterations in rhythm were also 
induced by paired electrical stimulation in 
man. Paroxysmal atrial tachycardia and atrial 
fibrillation were suppressed with paired stimuli 
delivered through a bipolar catheter in the 
right ventricle. Paired depolarizations may 
also be produced by allowing the normal, spon- 
taneous depolarization to act as the first depo- 
larization of the pair. This mode of stimulation 
has been referred to as "coupled pacing" and 
reduces the ventricular rate by approximately 
50%. 

We have termed the augmentation of myo- 
cardial performance produced by paired elec- 
trical stimulation electroaugmentation. The 
first method we employed for the demonstra- 
tion of electroaugmentation was measurement 
of myocardial contractile force. In eight open- 
chest dogs, in which right ventricular force 
was recorded with a myocardial strain gauge 
arch, paired electrical stimuli increased this 
variable to an average of 275 % of the level ob- 
served with single impulses delivered at the 
same rate. In other experiments, carried out on 
right heart bypass, a change from single to 
paired stimulation at an identical number of 
effective contractions per minute markedly im- 
proved the contractile state of the left ven- 
tricle. The peak rate of rise (dp/dt) of the left 
ventricular pressure rose, the duration of the 
systolic ejection period decreased, the mean 
and peak rates of ventricular ejection and 
stroke power increased, and the left ventricu- 



lar end-diastolic pressure usually fell. Catechol- 
amine depletion with reserpine, or blockade of 
beta-adrenergic receptors with pronethalol, did 
not diminish the degree of electroaugmentation 
produced by paired stimulation, indicating that 
mobilization of endogenous catecholamines is 
not responsible for this powerful inotropic re- 
sponse. 

Electroaugmentation also occurs in the in- 
tact human heart, as shown by investigating 
the effects of paired electrical stimulation in 17 
conscious patients. An increase in the rate of 
pressure development occurred, associated with 
a fall in ventricular end-diastolic pressure. . 
Studies on isolated human and cat papillary 
muscles showed how electroaugmentation 
shifts the myocardial force-velocity curve up- 
wards and to the right. It was also clear that 
paired stimulation exerts a positive inotropic 
effect on human ventricular muscle obtained 
from patients with congestive heart failure. 
Analysis of individual contractions indicates 
that summation of two contractions plays a 
small, but distinct role in electroaugmentation. 
No evidence was found to support the conten- 
tion that the extensibility of the muscle is 
changed by paired stimulation. Finally, it was 
shown that merely increasing the frequency of 
excitation does not account for the electroaug- 
mentation produced by paired electrical stimu- 
lation. 

The profound metabolic consequences of 
paired stimulation were observed in experi- 
ments in which the myocardial O2 consumption 
increased during the augmentation of left ven- 
tricular performance associated with paired 
stimulation, the increases averaging 35%. In 
all experiments the tension-time indices fell 
when the stimulation was changed from the 
single to the paired mode, the decreases aver- 
aging 14%. 

The effects of paired electrical stimulation 
on the heart are complex and clinicians will nat- 
urally wish to take advantage of the strongly 
positive inotropic action of this mode of stimu- 
lation for the treatment of cardiac failure. It is 
now clear that paired electrical stimulation is 
feasible in the human heart and it would seem 
possible to apply this technique of stimulation 
on a long-term basis — either by means of an 
implanted pacemaker delivering suitably 



132 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



spaced stimuli or by chronic endocardial stimu- 
lation. It is also evident that marked increases 
in the velocity of contraction and in the rate of 
pressure development may be expected to occur 
during paired electrical stimulation. However, 
the clinical benefits that may accrue from this 
speeding of the contractile process have not yet 
been defined. It has been disappointing that 
cardiac output has not risen consistently, even 
in the conscious patients with myocardial fail- 
ure. On the other hand, the decline of the ele- 
vated ventricular end-diastolic pressure may be 
of potential clinical benefit, particularly during 
exertion. 

Clinical. It is generally agreed that alter- 
ations in the rate of contraction constitute one 
of the most fundamental adaptive mechanisms 
available to the heart. Almost every major cir- 
culatory intervention, be it physiologic or phar- 
macologic, produces changes in heart rate. A 
technique was developed for controlling the 
heart rate in patients with normal atrioventric- 
ular conduction by means of an electrical 
pacemaker which stimulated the right atrium. 
When the heart rates were increased over a 
wide range, the cardiac indices remained essen- 
tially unchanged. The role of heart rate in the 
circulatory response to exercise was also exam- 
ined. When the heart rates were controlled at 
rates comparable to those achieved spontane- 
ously during exercise, it was observed that 
cardiac output rose normally with exercise and 
that this rise was accomplished entirely 
through an increase in the stroke volume. With 
isoproterenol infusion, when the heart rate was 
not permitted to rise, the increases in cardiac 
output were mediated through increases in the 
stroke volume. These studies indicate that 
heart rate plays little role in controlling the 
cardiac output at rest and that when metabolic 
demands are increased or the circulation is 
stimulated by catecholamines, the cardiac out- 
put can rise despite a fixed heart rate. 

Although it is acknowledged that ventricular 
end-diastolic size is an important determinant 
of ventricular performance, until recently it 
has not been possible to determine the effects 
of changes in heart rate per se on ventricular 
dimensions in man. At corrective cardiac oper- 
ation, roentgen-opague markers were sutured 
to the surfaces of one or both ventricles in nine 



patients. At postoperative cardiac catheteriza- 
tion, cineradiograms were exposed, and the 
distances between the markers were subse- 
quently measured. As the heart rate was in- 
creased by an average of 56 beats per minute, 
right and left ventricular end-diastolic and 
end-systolic dimensions decreased significantly. 
Calculations indicate that the average de- 
creases in external linear dimensions produced 
by elevation of heart rate in this investigation 
were equivalent to approximately 35 ml., or 
about one-half of a normal stroke volume. 

The effect of changing heart rate on the 
force of myocardial contraction has been in- 
vestigated in several animal species. While 
some species consistently have shown an in- 
crease in contractile force with increasing 
heart rate (the Bowditch "staircase" effect) a 
"reverse staircase" or decline in force with in- 
creasing rate has been demonstrated in others. 
Since the effect of changing heart rate on con- 
tractile force has not been determined directly 
in man, a study was undertaken to provide 
definitive information concerning this relation- 
ship. At the time of corrective cardiac surgery 
a Walton-Brodie strain gauge arch was sutured 
to the right ventricle of eight patients. While 
the Bowditch "staircase" effect does not appear 
to occur consistently in man, a "velocity 
treppe," i.e., an increase in the velocity of con- 
traction with increasing heart rate, does exist. 

The contractile properties of human heart 
muscle were studied in left ventricular papil- 
lary muscles excised from 19 patients at the 
time of prosthetic replacement of the mitral 
valve. Analysis of the isometric length-tension 
relations revealed that the peak of the active 
tension curve was reached when the muscle 
was stretched to an average length which ex- 
ceeded the initial length by approximately 
50%. Further increases in muscle length pro- 
duced a precipitous rise in resting tension, 
while active tension declined. An inverse rela- 
tion between afterload and initial velocity of 
shortening was observed in every muscle, ex- 
tending to human heart muscle the concept of 
force-velocity relations. When initial muscle 
length was increased, isometric tension was 
augmented but the maximum velocity of short- 
ening remained constant. The addition of stroph- 
anthidin or norepinephrine increased the 



NATIONAL HEART INSTITUTE 



133 



maximum velocity of shortening as well as the 
isometric tension, while increasing the fre- 
quency of contraction augmented the maximum 
velocity of shortening only. It is suggested that 
the contractile state of the human myocardium 
can be described by the force-velocity relation. 

An analysis of the series elastic component 
of the human myocardium indicated that this 
component was stretched an average of 8.4% 
during an isometric contraction, indicating 
that signiiicant shorteniing of contractile ele- 
ments occurs even in the absence of external 
muscle shortening. When the frequency of con- 
traction was increased, the duration of con- 
traction diminished, but the velocity of short- 
ening increased reciprocally. The resultant 
force developed, or extent of shortening, re- 
mained relatively unchanged. Thus, at any giv- 
en muscle length, the improved contractile 
state of the muscle resulting from increasing 
the frequency of contraction was reflected by 
the augmented power developed by the muscle, 
but was not evident in the external work per- 
formed. These studies on the contractile prop- 
erties of papillary muscles provide a frame- 
work for an analysis of the performance of the 
intact human myocardium in terms of funda- 
mental muscle mechanics. 

An investigation has also been carried out to 
determine whether or not the intact ventricle 
in conscious human subjects adheres to the bas- 
ic law of muscle contraction which had been 
elucidated in various experimental prepara- 
tions, and whether analysis of the force-veloc- 
ity relation can be useful in assessing the 
effects of various physiologic and pharmacolog- 
ic interventions on the human heart. Utilizing 
a cineradiographic technique, myocardial 
force-velocity relations were investigated in 
patients at the time of postoperative cardiac 
catheterization. The technique consisted of ex- 
posing cineradiograms at 30 frames per second 
and measuring the velocity of movement of 
roentgen-opaque markers which had been su- 
tured to the external surface of the ventricles, 
while simultaneously recording intraventricu- 
lar pressure. A beat-to-beat analysis of the 
force-velocity relation was then accomplished 
by measuring the velocity and the pressure at a 
constant length point in each contraction. 
When afterload was augmented with methoxa- 



mine or was decreased by impeding venous 
return with a balloon distended in the inferior 
vena cava, force and velocity varied inversely. 
In contrast, norepinephrine, isoproterenol, in- 
creasing heart rate by electrical stimulation, 
and paired electrical stimulation, all augment- 
ed velocity at any given pressure. Thus, in in- 
tact, conscious man, the heart displays the 
same reciprocal relation between velocity of 
shortening and generation of force observed in 
isolated papillary muscle, and a change in the 
contractile state of the human heart is mani- 
fested by a shift in the force-velocity relation. 

The response of the heart to exercise has 
also been analyzed by these methods. Because 
of the complex nature of the response of the 
heart to muscular exercise, it has been diflacult 
in intact human subjects to delineate directly 
the roles played by the Frank-Starling mecha- 
nism and by alterations in contractility. When 
exercise was performed while heart rate was 
kept constant by electrical stimulation, ventric- 
ular end-diastolic dimensions increased sig- 
nificantly. This augmentation in size contrasts 
with the diminution previously noted during 
exercise, when heart rate was allowed to rise. 
Therefore, the importance of the Frank-Starl- 
ing mechanism in the exercise response was 
made evident during electrical stimulation. 
Furthermore, the force-velocity curve shifted 
so that for any given pressure the velocity of 
shortening increased, indicating that myocar- 
dial contractility had improved. Thus, the nor- 
mal human heart can utilize several adaptive 
mechanisms during exercise. These include an 
elevation of heart rate, the Frank-Starling 
mechanism and an increased contractile state of 
the myocardium. It was then shown that fol- 
lowing beta-adrenergic blockade with propran- 
olol the augmentation of the contractile state of 
the heart normally seen during exercise is 
completely blocked. Nevertheless, an increase 
in cardiac output is still observed which is me- 
diated through the Frank-Starling mechanism. 

There has long been need for a practical 
method which would allow assessment of myo- 
cardial contractility in intact human subjects. 
Although the peak rate at which intraventricu- 
lar pressure rises (dp/dt) directly reflects the 
contractile state of the myocardium, this vari- 
able is also altered by changes in both initial 



134 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



size of the ventricle and by the afterload. Ex- 
periments in isolated muscle show that while 
dp/dt is dependent on both initial fiber length 
and afterload, the time from the onset of peak 
dp/dt (t-dp/dt) is constant for any one con- 
tractile state. However, inotropic agents which 
increase contractility characteristically shorten 
t-dp/dt concurrent with increasing the velocity 
of myocardial contraction. Therefore, an as- 
sessment was made of the usefulness of the 
combination of two measurements — dp/dt and 
t-dp/dt in the assessment of myocardial con- 
tractility in intact unanesthetized man. Ventric- 
ular dp/dt was continuously computed with 
an electronic differentiating circuilt and 
t-dp/dt calculated from high fidelity pressure 
recordings obtained at a fast paper speed in a 
large group of patients. With severe myocar- 
dial disease, the most prolonged t-dp/dt inter- 
vals were observed, with or without depression 
of dp/dt. In 15 patients, isoproterenol, ouabain 
or muscular exercise shortened t-dp/dt of both 
ventricles while dp/dt rose. These observations 
indicate that t-dp/dt extends the usefulness of 
dp/dt as an assessment of myocardial contract- 
ility and the combination of the two measure- 
ments provides a comprehensive analysis of 
ventricular performance in intact man. 

The function of the human left ventricle 
during exercise has previously been studied 
only indirectly, and in a relatively limited 
number of disorders involving the left side of 
the heart. Accordingly, an investigation was 
carried out involving the direct measurement 
of left ventricular pressure, measured directly 
by the transseptal, retrograde, or anterior per- 
cutaneous approaches. Left ventricular and 
aortic pressures, cardiac output and 0-, con- 
sumption were measured at rest and during ex- 
ercise on a bicycle ergometer. In patients with- 
out clinical or hemodynamic evidence of dis- 
ease involving the left ventricle, minimal 
changes in the left ventricular end-diastolic 
pressures were associated with increases in the 
cardiac index, stroke volume and stroke work, 
which were commensurate with the increases 
in the total O2 consumption. Using the limits 
established in these subjects, it has become pos- 
sible to characterize left ventricular function 
in a large number of patients with a variety of 
cardiac abnormalities. 



The precise mechanisms responsible for clo- 
sure of the atrioventricular valves under var- 
ious physiologic and pathologic circumstances 
have not yet been completely defined. During 
recent years, there has been particular interest 
in the role played by atrial contraction, an in- 
terest which stems largely from the reapprais- 
als of atrial function in experimental animals 
and man, and it has been suggested that the 
decline in atrial pressure during atrial relaxa- 
tion plays an important role in closure of the 
atrio-ventricular valves. In the course of diag- 
nostic studies in which selective angiocardio- 
grams with left ventricular injection were per- 
formed, we became impressed with the finding 
that the presence, or the development, of var- 
ious cardiac arrhythmias was not necessarily 
associated with perceptible mitral regurgita- 
tion. Accordingly, a review of 500 selective 
angiocardiograms in which the left ventricle 
was opacified was undertaken. This review 
yielded 43 studies in which the valve was com- 
petent in the absence of an appropriately timed 
atrial systole. From these observations, it has 
been concluded that a properly-timed atrial 
contraction is not ahvays essential for mitral 
valve closure in man. 

Action of Digitalis 

In spite of the widespread use of digitalis 
glycosides in clinical practice, neither the mech- 
anism of action of these drugs nor the rela- 
tionship between their inotropic and toxic prop- 
erties are fully understood. Radioautography 
and electronmicroscopy have been used in 
order to localize radioactive cardiac glycosides 
in order to determine on which cellular com- 
ponents these drugs act. Previous investiga- 
tions have indicated that in acutely digitalized 
hearts radioactive digoxin-H^ may be distrib- 
uted diffusely throughout the myocardium, as- 
sociated with the contractile substance itself 
without specific localization. Tritium labeled 
digoxin was administered to cats in graded 
amounts until electrocardiographic signs of 
glycoside toxicity appeared. Fifteen to twenty 
hours later, the animals were sacrificed and 
segments of ventricular myocardium were 
prepared for electronmicroscopic study. By ra- 
dioautographic techniques, digoxin-H= was lo- 



NATIONAL HEART INSTITUTE 



135 



calized primarily in the sarcoplasmic reticulum 
of the myocardium, especially in the area of 
the Z line of the sarcomere. Since this mem- 
1 brane system, which intimately surrounds the 
j sarcomere, is thought to be involved in excita- 
j tion-contraction coupling, it has been suggested 
i that cardiac glycosides may exert their salu- 
j tory action at this locus. These observations 
I are more in keeping with the physiological ac- 
tions of cardiac glycosides than previous views 
of glycoside localization. 

A considerable amount of confusion remains 
as to the effects of digitalis on the normal 
heart. The analysis of force-velocity relations 
in intact man receiving glycosides acutely has 
permitted a critical evaluation of this question. 
Using the methods for determining myocardial 
force-velocity relations described above, it has 
been possible to demonstrate that digitalis gly- 
cosides augment the contractile state of the 
myocardium with a shift in the force-velocity 
relations of the myocardium and a diminution 
in ventricular size, at a time when these drugs 
induced little or no changes in cardiac output. 

In order to provide more definitive informa- 
tion concerning the value of digitalis in pa- 
tients with abnonnal hemodynamic burdens, 
but without heart failure, a study was under- 
taken to determine whether digitoxin adminis- 
tered to rats before and after the production of 
hjrpertension by means of constriction of the 
abdominal aorta modifies the development of 
ventricular hypertrophy. When suitable correc- 
tions for variation in body weight were made, 
it was clear that aortic constriction resulted in 
the development of significant ventricular hy- 
pertrophy, which was reduced by digitalis 
treatment. The incidence of fatal congestive 
heart failure was also significantly lower in the 
animals which had been treated with glyco- 
sides. By reducing the development of hyper- 
trophy, digitoxin appears to permit the 
ventricles to sustain an excessive burden with 
less encroachment on this fundamental reserve 
mechanism. The results of this study would ap- 
pear to be of considerable significance when 
the prophylactic administration of digitalis is 
considered for patients who have not yet devel- 
oped heart failure, but who have an excessive 
load on the myocardium. 



Several investigators have proposed that the 
positive inotropic effect of digitalis on the 
heart is due solely, or in part, to norepineph- 
rine (NE) released from cardiac stores by the 
glycosides. This proposal is based on findings 
of a decreased inotropic effect of digitalis after 
depletion of cardiac stores of NE by reserpine, 
or after blockade of adrenergic receptors by 
methalide or dichloroisoproterenol. In these in- 
vestigations the drug used for sympathetic 
blockade of NE depletion has always been pres- 
ent during the study of digitalis effects and it 
is possible that some of the observations have 
been due to direct action of the drug rather 
than the interruption of sympathetic pathways 
to the heart. Accordingly the effects of the car- 
diac glycoside strophanthidin were studied in 
isolated papillary muscles obtained from nor- 
mal and chronically cardiac denervated cats. 
Papillary muscles from the latter animals were 
depleted of NE but exhibited a normal inotrop- 
ic response to strophanthidin. Accordingly, it 
is concluded that cardiac NE stores are not nec- 
essary for the mediation of the inotropic re- 
sponse to strophanthidin. 

The problem of digitalis intoxication re- 
mains of considerable practical importance. 
Indeed, the widespread use of potassium-deplet- 
ing diuretics appears to have increased the fre- 
quency of this condition. To determine whether 
suppression of the toxic manifestations of digi- 
talis by potassium affects the positive inotropic 
action of the drug, dogs were infused with oua- 
bain while right ventricular contractile force 
was measured with a strain gauge arch. When 
ventricular bigeminy or ventricular tachycar- 
dia occurred, KCl infusion was begun until 
sinus rhythm was restored. It was observed 
that a plateau in the development of contractile 
force during continuous ouabain infusion did 
not usually occur prior to the development of 
toxicity. It was also noted that suppression of 
the toxic manifestations by KCl permitted the 
further development of contractile force if the 
ouabain infusion were continued. The results 
of this study refute the contention that the 
maximum effect of digitalis glycosides occurs 
with doses much smaller than those producing 
toxicity, and emphasizes the need for the ad- 
ministration of doses of digitalis which are 
near toxicity, if the maximum positive inotrop- 



136 



ANNUAL REVIEW OF INTKAMURAL RESEARCH 



ic effect is to be attained. It also appears that 
the simultaneous administration of digitalis 
and KCl may improve the therapeutic/toxic ra- 
tio of the glycosides. 

As noted in an earlier section of this report, 
paired electrical stimulation of the heart is ca- 
pable of suppressing a variety of cardiac ar- 
rhythmias. An attempt was made to determine 
whether this technique would affect digitalis- 
induced arrhythmias. Two groups of closed- 
chest dogs received a constant ouabain infusion 
until death. Paired stimulation produced a 
regular and slower heart rate than that ob- 
served in the controls, but did not permit ad- 
ministration of a larger dose of ouabain before 
fatal ventricular fibrillation developed. In ani- 
mals in which ventricular tachycardia was pro- 
duced with a single dose of ouabain, paired 
stimulation suppressed the arrhythmia and 
maintained arterial pressure, and Avhen it was 
discontinued after two to six hours, the toxic 
effects of the glycoside had waned. The tech- 
nique of paired electrical stimulation may prove 
useful in clinical digitalis intoxication. 

Circulatory Control by the Autonomic Nervous 
System 

The activity of the autonomic nervous sys- 
tem is of fundamental importance in modulat- 
ing the alterations in circulatory function 
which occur during muscular exercise and a 
variety of other stressful situations. Accord- 
ingly a detailed comprehension of the operation 
of this system is essential to understanding 
cardiovascular control mechanisms. 

A depletion of sympathetic neurotransmitter 
had previously been demonstrated in this labo- 
ratory to occur in the heart in some patients 
with heart failure. Since stimulation of the 
cardiac sympathetic nerves exerts a positive 
inotropic influence upon the myocardium, it 
was of interest to determine if the degree of 
this depletion of norepinephrine (NE) was as- 
sociated with the alteration of myocardial 
function characteristic of the failing heart. 
The relation between cardiac NE concentration 
and the functional state of the myocardium 
was studied in left ventricular papillary 
muscles removed from patients with congestive 
heart failure at the time of mitral valve re- 



placement. Myocardial function was assessed 
by determining the maximum isometric active 
tension which the papillary muscle could devel- 
op in an in vit7-o system. A significant positive 
correlation was observed between the NE con- 
centration and the maximal developed force. It 
was concluded that NE depletion is associated 
with defective myocardial function, although 
no causal relationship between these two ab- 
normalities has been established. 

In other studies an attempt was made to as- 
sess sympathetic nervous activity in cardiac 
patients by measuring urinary excretion of NE 
and comparing this with the cardiac store of 
NE. It was observed that: 1) congestive heart 
failure is accompanied by an elevated activity 
of the sympathetic nervous system, as reflected 
in increased NE excretion; 2) sympathetic ac- 
tivity can be augmented further by the stress 
of operation; and 3) this sympathetic overac- 
tivity is often associated with a deficit of cardi- 
ac NE stores. The influence of this increased 
activity of the sympathetic nervous system with 
its associated augmented secretion of the neuro- 
transmitter hormone on the turnover rate of 
NE was evaluated by comparing the disappear- 
ance of radioactive NE from the urine in 5 
patients with normal activity of the sympathet- 
ic nervous system (urinary NE excretion av- 
eraging 26.0 /xg/day) and in 4 patients with 
heart failure and augmented sympathetic activ- 
ity and NE excretion averaging 73.6 /^g/day. 
DL-NE-7-H3 was administered and the decline 
of specific activity in urine determined for sev- 
eral days. The decay curves of specific activity 
in the urine were found to be similar in these 
two groups of patients. It is concluded that in- 
creased adrenergic activity does not appear to 
affect the turnover rate of the neurotrans- 
mitter substance and it is suggested that the 
rate of formation of NE is not controlled by 
the rate at which it is secreted from the adren- 
ergic neuron. It seems likely that NE synthesis 
is relatively constant and that during basal 
rates of secretion it is synthesized in amounts 
greater than required, the excessive amine be- 
ing removed by metabolism within the neurons. 
Since clinical heart failure is generally asso- 
ciated with cardiac hypertrophy, the possibility 
could not be excluded that the lowered NE con- 



NATIONAL HEART INSTITUTE 



137 



centration in the myocardium might represent 
simple dispersion of normal sympathetic nerve 
endings in an increased muscle mass, without a 
true depletion of total NE stores. Accordingly, 
this possibility was examined in experimental 
heart failure in two mammalian species. In the 
dog heart failure was produced by creating tri- 
cuspid insufficiency and pulmonic stenosis. 
When sacrificed six to eight weeks later, ascites 
and hepatic congestion were present in all ani- 
mals. Both concentration and total amounts of 
NE in both ventricles were strikingly reduced. 
The effects of tyramine, whose pharmacologic 
action is dependent on release of intraneuronal 
NE, were studied in isolated right ventricular 
papillary muscle preparations. The maximal 
increase in contractile force above control av- 
eraged 138% in muscles from control dogs and 
less than 20 % in muscles from dogs with heart 
failure. Similar studies using papillary muscles 
removed during mitral valve replacement from 
four patients with heart failure have also indi- 
cated a correlation between NE depletion and 
lack of tyramine response. From this study it 
was concluded that a true depletion of NE 
stores occurs in heart failure and that this 
depletion has striking pharmacologic conse- 
quences. 

In order to determine the nature of the de- 
fect in NE storage, NE retaining microsomal 
particles were isolated from cardiac tissue ob- 
tained from both normal and failing hearts of 
experimental animals. A defective ability to 
bind NE within this microsomal particle was 
found in the cardiac tissue from dogs with 
heart failure, and studies are in progress to 
characterize this abnormality. 

In order to study the physiologic conse- 
quences of the NE depletion in experimental 
heart failure, the response of heart rate and 
myocardial contractile force to stimulation of 
the right cardioaccelerator nerve was com- 
pared in a group of normal dogs and a group of 
animals with heart failure. It was observed 
that the response of animals with congestive 
heart failure to cardiac sympathetic nerve stim- 
ulation was significantly reduced below that 
of control animals, indicating that the reduced 
cardiac NE content observed in the failing 
heart actually interferes with sympathetic 
nerve function. Congestive heart failure was 



also produced in guinea pigs by supravalvular 
aortic constriction in order to elucidate the 
mechanism responsible for the changes in NE 
content observed. Significant reductions in NE 
stores in the ventricles occurred in the animals 
with aortic constriction and heart failure. The 
turnover of NE in the ventricles was not al- 
tered in heart failure but a defect of uptake 
and/or retention of NE was found to be pres- 
ent and may be responsible for the depletion 
of the cardiac NE stores. 

While it is clear that heart failure depresses 
the response of the myocardium to sympathetic 
nerve stimulation and to drugs which act by re- 
leasing local NE stores, there has been consid- 
erable debate concerning the manner in which 
NE stores affect the basic contractility of the 
myocardium. Thus, while the sympathetic nerv- 
ous system provides a mechanism for aug- 
menting basal cardiac contractility, it is not 
known if the cardiac stores of NE are neces- 
sary to establish normal baseline contractility 
and to maintain the potential for increasing 
contractility in response to positive inotropic 
interventions other than sympathetic stimula- 
tion. This question is of particular interest in 
view of the depletion of myocardial NE stores 
associated with congestive heart failure. Ac- 
cordingly, the contractility of papillary muscles 
obtained from cats with myocardial NE deple- 
tion produced by cardiac denervation or reser- 
pine treatment was studied. The maximum iso- 
tonic velocity of shortening as well as the max- 
imum isometric tension were not depressed in 
the NE depleted muscles. It was concluded that 
basal myocardial contractility and the potential 
for increasing it are not dependent on an intact 
cardiac store of NE. Preliminary studies indi- 
cate that these NE depleted papillary muscles 
are also supersensitive to exogenous NE. 

The role played by the adrenergic innerva- 
tion of the heart in the mediation of the car- 
diac response to muscular exercise was stud- 
ied by determining the effect of beta-adrener- 
gic receptor blockade on the hemodynamic re- 
sponses to maximal and submaximal exercise. 
A group of normal subjects were evaluated 
during high speed treadmill running, while 
measurements of cardiac output and heart rate 
were made before and after pharmacologic 
blockade of the stimulatory sympathetic nerves 



138 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



to the heart. Significant reductions in heart 
rate and cardiac output with identical levels of 
exercise following nerve blockade occurred. 
Less severe exercise was evaluated in six sub- 
jects with mild heart disease utilizing a fiber- 
optic catheter technique for measuring cardiac 
output continuously. These studies also showed 
a fall in heart rate and cardiac output after 
blockade of the sympathetic nerves to the 
heart. It has been concluded that the stimula- 
tory sympathetic nerves to the heart play an 
important role in mediating the heart's re- 
sponse to exertion. 

Considerable effort is being directed toward 
elucidating the precise mechanisms involved in 
the control of heart rate. In the past it has 
been believed that alterations in heart rate are 
brought about by simultaneous reciprocal 
changes in the sympathetic and parasympa- 
thetic influences exerted on the sino-atrial node. 
An investigation was therefore carried out to 
test the validity of this classical concept by 
defining the roles of the two segments of the 
autonomic nervous system in the regulation of 
heart rate. Augmenting arterial pressure above 
control levels with graded doses of phenyleph- 
rine always slowed heart rate strikingly, both 
in the dog and in man. Although complete sym- 
pathetic blockade with guanethidine or pron- 
ethalol did not significantly alter the degree of 
slowing, parasympathetic blockade with atro- 
pine or vagotomy essentially abolished this re- 
sponse. Conversely, lowering pressure with in- 
travenous nitroglycerin always raised heart 
rate in the control state, a response abolished 
by sympathetic blockade with guanethidine or 
pronethalol, but not by parasympathetic block- 
ade with vagotomy or atropine. Thus, when ar- 
terial pressure rises above control, the slowing 
of the heart rate is mediated by the parasym- 
pathetic nervous system, withdrawal of sym- 
pathetic activity playing no detectable role; 
when pressure falls below control levels, the 
elevation of heart rate is mediated primarily 
by the sympathetic nervous system. These find- 
ings are not consonant with the traditional 
concept of control of heart rate which predi- 
cates simultaneous reciprocal changes in activ- 
ity occurring in the two components of the 
autonomic nervous system. 



When normal atrioventricular (AV) conduc- 
tion takes place, the ventricular contraction 
rate is limited by the AV nodal system, which 
has been shown in experimental animals to 
have a longer refractory period than atrial or 
ventricular tissue. In spite of the fundamental 
importance of the refractory period of the AV 
nodal conduction system, this interval has not 
heretofore been measured in man, and little in- 
formation is available concerning its duration 
and the factors which modify it. A practical 
method for the measurement of the refractory 
period of the AV nodal system in conscious hu- 
man subjects was developed, normal basal val- 
ues for this period were established and the 
effects of tachycardia, exercise, atropine, and 
of various sympathomimetic drugs on its dura- 
tion were determined. A bipolar electrode cath- 
eter was positioned against the right atrial 
wall and stimulated the right atrium at a con- 
stant rate just above the sinus rate. Extra im- 
pulses were interposed between the regular 
stimuli ; the time intervals between these extra 
impulses and the preceding stimuli were then 
shortened progressively. The time interval at 
which AV conduction just failed was taken as 
the refractory period of the atrioventricular 
conduction system (AVRP). In 20 unanesthe- 
tized patients, studied in the basal state, the 
AVRP averaged 350 msec. Atrial tachycardia 
and the infusion of phenylephrine prolonged 
the AVRP while atropine, isoproterenol, and 
exercise shortened it. Thus, in any given pa- 
tient the AVRP is affected profoundly by sym- 
pathetic and parasympathetic stimuli, and 
measurement of the AVRP permits quantifica- 
tion of these effects. 

The reflex response of the arterial and ven- 
ous beds of the forearm to changes in posture 
and cold stimulation were determined in a 
group of patients with familial dysautonomia, 
and in a group of normal subjects, with a 
plethysmographic technique. Tilting to a head- 
up position resulted in no augmentation of ar- 
terial or venous tone in patients with dysauton- 
omia, but resulted in striking elevations of 
both of these variables in normal subjects. Sim- 
ilarly, cold stimulation did not elicit an increase 
in arterial and venous tone in the dysautonomic 
patients, a response which was observed 
consistently in the normal subjects. The 



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aosence of these vasomotor reflexes provides 
an explanation for the orthostatic syncope 
which is an important clinical feature of famil- 
ial dysautonomia. 

Regional Circulations 

Coronary Circulation 

Previous experiments have suggested that an 
intrinsic adrenergic mechanism for vasocon- 
striction exists in the coronary vascular bed. 
Evidence that the coronary vascular bed also 
contains an intrinsic adrenergic mechanism for 
vasodilatation (a beta-receptor system) was 
obtained by studying the response of canine cor- 
onary blood vessels to isoproterenol. In intact 
animals and in isolated potassium-arrested 
hearts, this drug always decreased coronary 
vascular resistance. The resistance change 
could be blocked by pronethalol and did not 
depend upon increased myocardial metabolism 
or a decreased level of myocardial oxygenation. 

Limb Circulation 

The vascular dynamics of the forearm were 
determined at rest and after several interven- 
tions in normal subjects and in patients with 
heart failure. At rest, forearm blood flow var- 
ied directly with cardiac output, forearm re- 
sistance varied directly with total resistance, 
venous tone varied directly with forearm re- 
sistance, and indirectly with cardiac output. 
Patients with heart failure always had lower 
values for forearm flow and higher values for 
forearm resistance and venous tone than did 
normal subjects. The cold pressor test and leg 
exercise resulted in an excessive elevation of 
forearm resistance and venous tone, and leg 
raising resulted in a reduction of forearm 
blood flow in patients with failure and an ele- 
vation in normal subjects. Reactive hyperemia 
was reduced in the heart failure patients. 

The effects of stimulation of the atria, by 
electrically pacing the right atrium with a 
catheter electrode and thereby increasing the 
atrial contraction rate, on the vascular dyna- 
mics of the forearm, and on the total peripher- 
al vascular resistance, were studied. Forearm 
blood flow became elevated, and arterial and 
venodilatation occurred, while the total system- 
ic vascular resistance generally increased. 



From this investigation it is postulated that 
there are receptors in the walls of the atria, the 
stimulation of which are capable of producing 
vasodilatation of the normal human forearm. 

It is now well established that the endoge- 
nous nonapeptide bradykinin has important 
effects on the circulatory system, and, in addi- 
tion, may play a role in many physiologic and 
pathologic clinical conditions. The effects of the 
intravenous injection of synthetic bradykinin 
on the arterial and venous beds of normal hu- 
man subjects were evaluated by a plethysmo- 
graphic technique. Bradykinin produced a de- 
crease in systemic arterial pressure, an increase 
in forearm blood flow, therefore, result- 
ing in arterial dilatation. Following a brief 
period of venoconstriction, venodilatation oc- 
curred. The former was abolished by adrener- 
gic blockade with guanethidine. From this 
investigation, it Avas concluded that (a) the 
venoconstriction is a compensatory reflex elicit- 
ed during the fall in arterial pressure, and (b) 
that the prime effect of bradykinin is one of 
arterial and venodilatation. 

In order to determine if the possible periph- 
eral circulatory actions of the nitrite drugs 
might play a role in their important therapeu- 
tic effects, the responses of the arterial and 
venous beds of the forearm to sublingual nitro- 
glycerin and inhaled amyl nitrite were deter- 
mined in a group of normal subjects. Nitroglyc- 
erin resulted in dilatation of both the arterial 
and venous beds, while amyl nitrite resulted in 
dilatation of the arterial bed but constriction 
of the venous bed. This latter effect was 
blocked by the antiadrenergic agent guanethi- 
dine, and thus was felt to be a reflex elicited by 
the profound fall of arterial pressure. In order 
to determine whether the peripheral actions of 
nitroglycerin reduced heart size, small silver 
tantalum markers were sutured to one or both 
ventricles of 11 patients at the time of cardiac 
operations. Following recovery from operation, 
the distance between markers on cineradio- 
grams exposed at 30 frames/sec. was measured 
before and after 0.6 mg. of nitroglycerin. In all 
patients nitroglycerin decreased end-diastolic 
and end-systolic dimensions within 2 to 6 
minutes, the changes in end-diastolic vol- 
ume approximating 30% of the stroke vol- 
ume. Systolic excursions decreased by an aver- 



140 



ANNUAL REVIEW OF INTRAMUKAL RESEARCH 



age of 13.3% for the right ventricle and 9.0% 
for the left ventricle. From these studies, it is 
clear that nitroglycerin reduces ventricular di- 
mensions in man, and in view of the known re- 
lationships between ventricular volume, wall 
tension, and myocardial oxygen consumption, 
favorably influences the balance between myo- 
cardial O2 availability and 0, requirements, 
thus explaining, at least in part, its effective- 
ness in angina pectoris. 

Pulmonary Circulation 

The control of the pulmonary circulation has 
always been difficult to study, since before an 
effect on the pulmonary circulation can be as- 
cribed to any specific stimulus acting on the 
pulmonary vessels, it must be shown to act in- 
dependently of changes in the systemic circula- 
tion, body position, and phase of respiration. A 
new technique was devised for assessing 
regional pulmonary blood flow in man by ra- 
dioisotope scintillation scanning of the distri- 
bution of the lungs of intravenously injected 
radioactive macroaggregated albumin (MAA). 
Densitometric quantification of the distribution 
of MAA in the lung field following injection of 
acetylcholine into a single pulmonary artery 
revealed that acetylcholine exerted a vasodila- 
tory effect. A systematic analysis of the effects 
of a variety of drugs on the pulmonary circula- 
tion utilizing this technique is planned. 

Improvement of Cardiac Diagnostic Methods 

During the past two decades there has been 
steady improvement in the instrumentation for 
recording information obtained at cardiac 
catheterization. With the development of effec- 
tive methods for image intensification, the im- 
proved fluoroscopic image greatly facilitated 
catheter manipulation, and cineradiography 
and cineangiocardiography became practical 
clinical techniques. Concurrently, the perfec- 
tion of catheter-tip manometers, catheter-tip 
microphones and oximeters has expanded and 
refined the data that can be recorded oscillo- 
graphically. However, it has not been conven- 
ient to make precise temporal correlations be- 
tween the radiographic image recorded on the 
cine film, and data displayed oscillographically, 
such as the electrocardiogram, and the intra- 



cardiac pressure pulses. A device termed the 
"Cinetrace" was developed in order to superim- 
pose the data displayed on the cathode ray os- 
cilloscope onto one corner of the cine film. 
Areas of obstruction to blood flow have been 
localized precisely by moving the catheter 
across the obstruction and recording the posi- 
tion of the catheter tip at the instant of pres- 
sure change. The technique has been used in 
the diagnosis and localization of valvular, su- 
pravalvular and subvalvular obstruction to 
right and left ventricular outflow. 

The clinical applications of an experimental 
in vivo oximeter system employing a fiberoptic 
catheter have also been investigated. The in- 
strument permits, for the first time, the direct 
measurement of blood 0. saturation at the 
catheter tip, without the withdrawal of blood. 
By a change of optical filters, the instrument 
can be made linearly responsive to indocyanine 
green dye concentration. An electrode at the 
catheter tip permits the recording of the intra- 
cardiac electrocardiogram and facilitates pre- 
cise localization of the catheter. The instru- 
ment has been found to be useful in facilitating 
rapid and thorough catheterization studies in 
patients with left-to-right shunts; it has made 
possible a study of the phasic changes in pul- 
monary arterial Oj saturation during respira- 
tion and during the Valsalva maneuver ; it has 
permitted continuous measurement of pulmo- 
nary arterial O2 saturation before, during, and 
after exercise in the supine or upright posi- 
tions, and it has permitted a study of the 
effects of pharmacologic interventions upon 
this variable. 

The inability to excrete ingested Na is one of 
the most fundamental abnormalities which 
characterizes the congestive heart failure state. 
Indeed, most of the symptoms and physical 
signs which occur in heart failure — exertional 
dyspnea, orthopnea, edema, venous distention, 
hepatomegaly and ascites — result in large meas- 
ure from retention of Na and water. A variety 
of methods is now available for quantifying 
certain aspects of circulatory function in man. 
None of these approaches, however, provides 
information concerning the patient's ability to 
excrete ingested Na. Accordingly, a simple oral 
Na tolerance test, designed to detect and quan- 
tify abnormalities of sodium excretion was de- 



NATIONAL HEART INSTITUTE 



141 



veloped. Following a 4-day period during 
which daily Na intake was limited to 10 mEq., 
80 mEq. Na was administered daily for 4 days 
and 150 mEq. for another 4 days. The total 
urinary Na excretion during the 8 day test pe- 
riod was determined. Thirteen normal subjects 
excreted between 550 and 734 mEq. Na. Of 41 
patients with heart disease, 31 excreted sub- 
normal amounts of Na, between 10 and 550 
mEq. and only 10 excreted normal quantities. 
There was no correlation between the impair- 
ment of Na excretion, as estimated by the Na 
tolerance test, and the etiology of the heart dis- 
ease, the glomerular filtration rate, or any he- 
modynamic variable. Striking improvement in 
N^ tolerance followed corrective cardiac opera- 
tions. The Na tolerance test has been found 
particularly useful in the evaluation of patients 
in whom clinical examination and hemodynam- 
ic studies gave no conclusive evidence as to 
the presence or absence of congestive heart 
failure. 

Clinical Studies 

The clinical findings in 64 patients with idio- 
pathic hypertrophic subaortic stenosis (IHSS) 
were analyzed and correlated with the physio- 
logic abnormalities in this disease. The hemo- 
dynamic response of the left ventricular 
outflow tract was determined in a group of pa- 
tients with IHSS during change in body posi- 
tion. It was observed that leg raising with the 
patient supine, or tilting to a head-down posi- 
tion reduced the outflow gradient and in- 
creased the effective outflow orifice area. In 
contrast, tilting to a head-up position resulted 
in an increase in the intraventricular pressure 
gradient and a reduction of the outflow orifice. 
These changes are probably related to the pro- 
duction of symptoms in these patients and may 
be responsible for the orthostatic syncope that 
is often observed. 

The infusion of the cardiotonic agent isopro- 
terenol in patients with different forms of ob- 
struction to right ventricular outflow resulted in 
an increase of the systolic pressure gradient 
across the pulmonary outflow tract. A diminu- 
tion of the effective outflow orifice occurred in 
patients with idiopathic hypertrophic subpul- 
monic stenosis and in postoperative patients 



with secondary subvalvular stenosis who had 
undergone successful surgical relief of valvular 
stenosis. In contrast, however, no decrease in 
the pulmonic outflow area was observed in pa- 
tients with valvular pulmonic stenosis. These 
observations indicate that muscular obstruc- 
tion to right ventricular outflow is dynamic, 
that it can vary in severity depending on the 
heart's catecholamine background, and that the 
response to infusion of isoproterenol can be 
used to distinguish muscular from fixed ob- 
struction. The response to isoproterenol thus 
provides a useful test for assessing the nature 
of obstruction to right ventricular outflow. 

The development of an elevation of the pul- 
monary vascular resistance is a common com- 
plication of severe mitral valve disease, and 
this abnormality has been attributed to a com- 
bination of obliterative changes in the pulmo- 
nary vascular bed and pulmonary vaso- 
constriction In many patients the elevation of 
pulmonary vascular resistance is extreme and 
constitutes the major hemodynamic burden 
faced by the right ventricle. In view of these 
considerations, determination of the extent to 
which the abnormalities in the pulmonary cir- 
culation can be reversed by surgical correction 
of the valvular lesion is critically important, 
particularly when operation is contemplated in 
a patient with extreme pulmonary hyperten- 
sion. To investigate this question, pulmonary 
vascular dynamics were evaluated both pre- 
and post-operatively in 31 patients with mitral 
valve disease in whom the valve was replaced 
with a Starr-Edwards prosthesis, and whose 
pulmonary arterial systolic pressures were 50 
mm. Hg prior to operation. The average pul- 
monary arterial or right ventricular systolic 
pressures declined from a preoperative level 
of 75 mm. Hq to 39 mm. Hg. The gradient 
across the pulmonary vascular bed decreased 
while the cardiac output rose and the average 
calculated pulmonary vascular resistance fell 
from 679 dynes-sec-cm '^ preoperatively to 249 
dynes-sec-cm"" postoperatively. It was con- 
cluded that valve deplacement generally re- 
sults in striking reductions in pulmonary vas- 
cular pressures and resistances in patients 
with serious mitral valve disease and pulmo- 
nary hypertension. 



142 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



A rational decision concerning operation in 
patients with congenital heart disease and pul- 
monary hypertension is dependent upon an un- 
derstanding of the natural history of the 
disease. Accordingly, serial clinical and 
hemodynamic findings in patients with sys- 
temic-pulmonary communications and pul- 
monary vascular obstruction are being analyzed 
in an attempt to understand the natural history 
of the Eisenmenger reaction. Twenty-two pa- 
tients with isolated ventricular septal defects 
and patent ductus arteriosus have thus far 
been studied. Elevated pulmonary vascular 
resistance may persist from birth in a large 
number of these patients. Symptoms often be- 
gin during the second and third decades of life 
and survival to the fourth and fifth decades 
may be anticipated in the absence of surgical 
therapy. 

Section of Clinical Biophysics 

It is the purpose of this section to engage in 
basic experimental studies of the cardiovascu- 
lar and pulmonary systems with an ultimate 
objective of establishing realistic and useful 
mathematical models of these systems. This 
implies an active laboratory program with 
strong computer support. The current activi- 
ties of this section may be summarized arbitrar- 
ily under four headings: Pulmonary Mechan- 
ics, Pathogenesis of Emphysema, Myocardial 
Mechanics and Vascular Mechanics. 

Pulmonary Mechanics: Studies have contin- 
ued in an attempt to define further the physical 
properties of the pulmonary system. These stud- 
ies have yielded anatomical and rheologic in- 
formation which has permitted us to develop 
what appears to be a very promising mathemat- 
ical model of the lung. In view of the extremely 
complex nature of the lung our approach to this 
project necessarily has been modular. That is, 
having identified and isolated each of the per- 
tinent components of the system, study of the 
individual parts then can be pursued relatively 
independently. 

In essence this model consists of an ensemble 
of expansile airspaces that ventilate through 
an arborized collapsible conduit system, the fi- 
nal confluence of which is the trachea. On the 
outer surface of this conduit system, there is a 
distribution of stress which is determined by 



the intrathoracic pressure and forces generat- 
ed within the surrounding medium. On the 
inner or intraluminal surface of this conduit 
system there is another stress distribution 
which is related to the physical properties of 
the gas, the flow, and the instantaneous dimen- 
sions of the conduit system. These dimensions, 
in turn, are related to the difference between 
these two stress distributions. With this model 
in mind the following questions arise. What is 
the nature of the extramural stress? What is 
the nature of the intraluminal stress ? 

The extramural stress on the conduit system 
is the algebraic sum of the pressure in the con- 
tiguous airspaces plus the stresses generated in 
the surrounding tissues. A number of indirect 
techniques have been developed for evaluation 
of the airspace pressures; however, the stress 
in the surrounding tissues remains to be eval- 
uated. If it can be determined that the vis- 
ceral pleura plays an essentially negligible role 
in the visco-elastic behavior of the total lung 
system, then measurement of the "transpleural 
pressure" should reflect these tissue stresses. 
Therefore, a series of studies have been carried 
out to evaluate the elastic properties of the vis- 
ceral pleura. A transverse diameter of exposed 
lung was continuously recorded with a spe- 
cially designed electrical recording caliper. 
Tracheal pressure was monitored simultane- 
ously with pressures from the distal points in 
the lung near the region to which the legs of 
the recording caliper were attached. Pressure 
vs. dimensional relations could then be re- 
corded continuously during various imposed re- 
spiratory maneuvers. Following measurement 
of control pressure-diameter relationships, the 
pleura was slit circumferentially in planes nor- 
mal to the lung diameter between the legs of 
the caliper to relieve circumferential tensions 
in the pleura itself. Pressure-diameter relation- 
ships were determined again following this 
procedure. It was found that the pleura did not 
affect the pressure-diameter relationship of the 
lung. Although there are minor rheologic 
questions which must be pursued in this area, 
the important point is that the pleura does not 
play a major role in determining the stress- 
strain relationships of the lung. Thus, to a first 
approximation one many consider the trans- 



NATIONAL HEART INSTITUTE 



143 



pleural stress to be identical to the extramural 
stress on a conduit system. 

The distribution of stress on the intralumin- 
al surface of the conduit system is considerably 
more complex. This stress distribution depends 
on the complicated pressure-flow relationships 
that occur in nonuniform conduit systems. This 
problem occupies the frontiers of current day 
aerodynamics. There are no mathematical mod- 
els which describe the pressure-flow relation- 
ships within nonuniform boundaries such as 
those of the bronchial tree. Therefore, it be- 
comes important to establish empirical rela- 
tions describing pressure-flow behavior in phys- 
ical models of the pulmonary tree. Glass and 
plastic tubes scaled to various dimensions simi- 
lar to those in the pulmonary tree were fash- 
ioned in various geometric configurations. 
The pressure distribution was measured along 
the length of these conduits at different flow 
rates using gases of known viscosities and den- 
sities. Pressures were sampled with various 
specially designed exploratory probes. The 
pressure, the flow, and the site of pressure 
measurement were continuously recorded on 
electromagnetic tape for A/D conversion and 
digital computer processing. A family of equa- 
tions representing approximate solutions to the 
Navier-Stokes equation was screened with 
these data. Two of these solutions were found 
to represent the aerodynamics of these nonuni- 
form conduits satisfactorily. The parameters 
of these equations may be expressed in terms 
of the physical properties of the gas as well as 
certain dimensions of the conduit system. 

Armed with the results of the foregoing two 
series of studies, i.e. the nature of the extramu- 
ral stress and intramural stress distributions, 
it was possible to challenge the proposed 
mathematical model of the lung. This was ap- 
proached in two ways : study of physical models 
and study of human data. 

Physical models were constructed to repre- 
sent various segments of the over-all lung mod- 
el. Various conduits were chosen that had prop- 
erties very similar to those of bronchial seg- 
ments. The pressure-area relationships to these 
conduits and their dimensions were carefully 
measured. Then the pressure-flow behavior of 
these systems was studied under carefully con- 
trolled conditions of transmural stress using a 



variety of special transducers and recording 
techniques. Both liquid and gas mixtures were 
studied in these systems so that a wide range 
of densities and viscosities could be examined. 
These data were then analyzed in a nondimen- 
sional format such that tubes of various sizes 
as well as subsequent human data could be 
viewed in a similar coordinate system. The abil- 
ity of the mathematical model to predict the 
behavior of the physical models was extremely 
encouraging. Moreover, when the nondimen- 
sionalized data from the human studies were 
plotted on the family of predicted curves, 
equally good agreement was found. For ex- 
ample, data taken from maximum expiratory 
flow- volume curves of human subjects breath- 
ing various gas mixtures fell very close to the 
predicted curve for the model having bronchi 
of 1.5 mm I.D. 

The agreement between the predicted behav- 
ior and observation has provided a good level 
of confidence in the mathematical model. These 
studies have been extremely informative in 
shedding light on heretofore unexplained ob- 
servations. For example, we now have clear in- 
sight into the physical events associated with 
mechanical autoregulation of flow both in the 
pulmonary bronchial tree and in various other 
biological flow systems. It is now possible to in- 
terpret such terms as "vascular resistance" 
particularly in the pulmonary circulation more 
explicitly in terms of precise physical events. 
Moi^eover, the mechanisms underlying the var- 
ious paradoxical observations regarding ven- 
ous return to the chest, renal and urine flow, 
and cerebral blood flow can be explained in 
unequivocal physical terms. 

Although the foregoing should be of interest 
to the general scientific community, the most 
immediate interest from our point of view is 
the possibility that we will be able to develop a 
realistic mathematical model of the total lung 
system which can be used in the analysis and 
interpretation of relatively easily obtained clin- 
ical measurements of pulmonary function such 
as the maximum expiratory flow volume curve. 

Pulmonary Emphysema 

Two of the many obstacles in advancing our 
understanding of pulmonary emphysema have 



144 



ANNUAL REVIEW OF INTKAMURAL RESEARCH 



been the lack of an experimental animal and 
our inability to recognize the early pathologic 
and physiologic lesion of the disease. There is 
one suggestion in the literature that rabbits de- 
velop pulmonary emphysema spontaneously. 
Studies in collaboration with the Pathology 
Branch of the National Cancer Institute are 
under way to attempt to reproduce this obser- 
vation. In the meantime studies have been done 
to develop methods of evaluating pulmonary 
mechanics in the rabbit since standard methods 
for these measurements in a laboratory animal 
do not exist. 

A small body plethysmograph has been de- 
signed and fabricated in which instruments 
have been developed for the accurate measure- 
ment of instantaneous respiratory flow, vol- 
ume, and pressure. The plethysmograph itself 
consists of a rigid plastic chamber in which it 
is possible to ventilate anesthetized animals ar- 
tificially. A positive displacement electrical re- 
cording spirometer continuously records the 
volume changes of the animal. Instantaneous 
flows are measured by a specially designed lin- 
ear flow resistance device. These instruments 
were found to be linear, free of hysteresis, and 
to have a uniform dynamic response through 
20 cps. Two large groups of rabbits, one group 
four months old and the other in excess of two 
years, have been studied with this device. A 
detailed analysis of the pressure-flow-volume 
data from these experiments has not been car- 
ried out as yet. However, a preliminary crude 
analysis indicates a significant diff'erences be- 
tween the young and old rabbits. If the sponta- 
neous development of pulmonary emphysema in 
these rabbits can be confirmed both physiologi- 
cally and pathologically, this will represent a 
inajor advance in our knowledge. Both longitu- 
dinal and transverse studies of normal rabbit 
populations will be carried out employing these 
newly developed techniques. Following this, var- 
ious experimental interventions will be ex- 
plored. The first of these will be to examine the 
effect of various noxious gases and the second 
to examine the effect of papain an agent known 
to dissolve the cartilaginous support of the 
bronchial tree. Hopefully, the mathematical 
model of the lung described earlier will have 
been validated by this time so that the data 
from these studies may be processed in accord- 



ance with the model so that the observed 
changes may be interpreted in terms of altered 
physical properties and dimensions of the sys- 
tems. 

Myocardial Mechanics 

The development of a realistic mathematical 
model of the heart poses a number of formi- 
dable questions. In building this model (just as 
in building the lung model) , it is necessary to 
view the behavior of the individual components 
and then the behavior of the entire ensemble. 
Studies that bear on these two objectives have 
been designed. The behavior of the components 
of the system has been explored with various 
in vitro papillary muscle preparations. To date, ■ 
both cat and rabbit muscle preparations have 
been developed to permit a precise and contin- 
uous measurement of instantaneous muscle 
length, shortening rate and developed tension 
as a function of time under carefully controlled 
conditions. The preliminary results of this 
study indicate that a force-velocity relationship 
similar to that for skeletal muscle may exist in 
the initial phases of the contraction ; however, 
during the latter half of contraction, velocity 
appears to approach some limiting value, be- 
coming relatively independent of either length 
or load. Various mathematical models are be- 
ing developed to describe this behavior which 
are in some respect similar to Hill's "charac- 
teristic equation" for skeletal muscle but, in 
other ways, quite different. 

The integrated behavior of an ensemble of 
these muscle fibers will depend on the pattern 
of excitation, the over-all geometry of the heart 
wall, the stresses that exist across the wall, and 
finally on the distribution and orientation of 
the component fibers within the wall. Since the 
duration of contraction is quite long compared 
to the time for spread of excitation in a normal 
heart, the pattern of excitation has been as- 
sumed to be a "second order" effect, at least for 
the time being. Therefore, the major unresolved 
questions relate to the geometry of the heart 
wall and to the distribution and orientation of 
fibers within it. 

As an initial approach to the measurement 
of the gross geometry of the heart, a prepara- 
tion was designed such that the inflow to the 



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145 



left ventricle could be rapidly switched from 
blood to a large volume of liquid plastic mate- 
rial. The separation of a number of points on 
the ventricular surface was recorded contin- 
uously using electrical calipers during a control 
period and then during the sequence of events 
following the inflow of the liquid plastic. The 
heart finally comes to rest, filled with this 
plastic material which rapidly hardens. To the 
extent that the recordings of surface geometry 
remain in the same region after this procedure 
as they did during the control period, it is as- 
sumed that the "fixed" heart has essentially the 
geometry that it has during the normal beating 
state. Various dimensions are then measured 
and recorded. The gross shape of the myocar- 
dium has been found to vary somewhere be- 
tween a cylindrical and an ellipsoidal configura- 
tion. 

Following these gross studies the heart was 
fixed, sectioned serially, and examined micro- 
scopically. The sections were taken in mutually 
perpendicular planes so that the orientation 
and number of fibers at each position could be 
determined. The approximate distribution of 
fibers in the ventricle is as follows: The inner 
40% of the wall has fibers running in the axial 
direction, the middle 50% has fibers running in 
a circumferential direction and the outer 10% 
again has fibers in the axial direction. Al- 
though there is considei-able variability in some 
instances, these patterns are generally well 
oriented and easily recognizable. These findings 
are consistent with the physiologic studies de- 
scribed in last year's Annual Report in which it 
was noted that the heart appears to contract 
with "two degrees of freedom." The implica- 
tions of these anatomical and physiologic meas- 
urements are of obvious significance in the 
mechanics of contraction. 

Vascular Mechanics 

Success in providing a model of the vascular 
bed depends upon determining valid mathema- 
tical descriptions of the component vascular 
segments. Only when this has been accom- 
plished can one turn to description of the inte- 
grated behavior of the system. Therefore, the 
major effort of this section has been in deter- 
mining the properties of these small "building 



blocks" of the system. The mechanical behavior 
of a small vascular segment depends on the 
properties of the vessel wall as well as the un- 
derlying flow. Under controlled conditions 
these two quantities may be evaluated independ- 
ently. Therefore, one set of studies was de- 
signed to examine the laws governing blood 
flow (the laws of fluid motion) and a separate 
set of studies was designed to explore the laws 
governing the geometry and physical behavior 
of the vessel walls (the boundary equations of 
the system) . 

Fluid motion is determined by the physical 
properties of the liquid and the pressure gra- 
dient. This relationship is expressed formally 
by the Navier-Stokes equations which are com- 
plicated nonlinear partial differential equa- 
tions. Simplified solutions to these equations 
have been derived. These mathematical models 
were then tested experimentally both in var- 
ious flow generating devices as well as in vitro 
dogs True flow in a pulsating vessel was esti- 
mated indirectly using two electromagnetic 
flowmetering systems. A pressure gradient was 
measured simultaneously and used as the 
"forcing function" for the mathematical model 
of flow. The resulting computed flow was then 
compared with the estimated flow from the 
electromagnetic flowmeter systems. The stand- 
ard deviation of the instantaneous coordinate 
values of the computed flow compared to the 
"true" flow averaged about ±7 cc per sec. 

Mathematical models of the vascular fluid 
boundaries have been developed using data ac- 
quired from a number of specially designed 
transducers which record continuously the in- 
stantaneous diameter-length relationships and 
the longitudinal tethering of various arterial 
segments. Longitudinal and circumferential 
stresses and strains were studied to evaluate 
the visco-elastic properties of the aorta in the 
three principle dimensions of the system, 
(longitudinal, radial, and circumferential). 
Longitudinal constraints of the aorta were stud- 
ied quantitatively by studying the forces asso- 
ciated with both transient and sinusoidal longi- 
tudinal displacements of the vessel segments. 
The major findings of these studies are that 1) 
the distensibility of the aorta decreases pro- 
gressively along its length from root to bifur- 
cation. 2) At any point the elastic properties 



146 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



are different in the three directions, i.e. the 
vessel wall in anisotropic. 3) The aorta is con- 
strained from longitudinal motion by an "iner- 
tio-visco-elastic body." The mathematical mod- 
el of this vascular tethering phenomenon has 
been formulated to include the usual behavior 
of "real" tissues such as stress relaxation, vis- 
cosity, hysteresis, etc. This model has shown 
surprising agreement with observation. 

The foregoing studies clarify the relation- 
ships between instantaneous flows, pressure, 
and vessel dimensions in the circulatory sys- 
tems of animals. This is of considerable theoret- 
ical importance in the field of rheology, fluid 
mechanics, and circulatory dynamics. It is also 
of practical importance to the clinical physiolo- 
gist who can apply these principles to the meas- 
urement of instantaneous blood flow from 
more easily obtained pressure information. 
These studies are being pursued in an effort to 
establish progressively more comprehensive 
models of the circulatory system thereby ex- 
tending our insight into the mechanisms of 
normal and "abnormal" circulatory dynamics. 

SURGERY BRANCH 

The research projects of the Surgery Branch 
have, as in past years, largely centered around 
studies of normal and abnormal circulatory 
physiology, particularly in relation to the 
effects of surgical treatment in patients with 
congenital or acquired cardiac malformations. 
Studies have been made in both the laboratory 
and operating room, but studies in man have 
been made with increasing frequency utilizing 
the system for data acquisition provided in the 
surgical wing. 

More than half of the patients referred to 
the Surgery Branch now require operations in- 
volving the insertion of prosthetic cardiac 
valves, and a significant proportion of the work 
of the unit has been allied to this general prob- 
lem. A previous report has described the unique 
opportunity provided at operation for the 
measurement of the magnitudes of forward 
and regurgitant blood flow in patients with 
aortic regurgitation. These studies have been 
extended to include the circulatory dynamics in 
aortic regurgitation not only under basal con- 
ditions, but also during various interventions. 



While instantaneous aortic flow is measured 
with a flowmeter on the ascending aorta, the 
heart rate has been controlled and altered by 
electrical pacing. Contrary to general belief, it 
has not been found that a rapid heart rate is 
beneficial, in terms of net forward blood flow, 
to patients with aortic regurgitation. In all pa- 
tients studied, maximum forward flow oc- 
curred at a rate between 80 and 100 per min- 
ute. Further investigations are being made 
concerning the effects on regurgitant flow of 
inotropic agents, alterations in circulating 
blood volume, and of increased and decreased 
peripheral arterial resistance. 

In most institutions the use of prosthetic 
cardiac valves has been complicated by the de- 
velopment of bacterial endocarditis in the early 
postoperative period. This has not occurred in 
this Clinic, but three patients developed bacte- 
rial endocarditis in the late postoperative 
period, and in each the infection was fatal. 
This experience has led to detailed re-evalua- 
tion of antiobiotic prophylaxis in patients with 
prosthetic valves, particularly when dental or 
operative interventions are necessary. 

Patients with prosthetic valves in the mitral 
position are now maintained indefinitely on an- 
ticoagulation, but, even so, systemic emboli re- 
main a constant threat to them. When the 
prosthesis is in the tricuspid position, clot for- 
mation and embolization are even more fre- 
quent. It is becoming evident that the tendency 
to thrombus formation varies from patient to 
patient, and may be measured by determina- 
tions of platelet adhesiveness. Adhesiveness is 
measured by determining the fraction of the 
platelets extracted from blood as it is passed 
through a column of fine glass beads. Prelimi- 
nary information indicates that this study is of 
prognostic significance, and also that adhesive- 
ness can be greatly reduced by the adminis- 
tration of low molecular weight dextran or the 
maintenance of a low hematocrit. Experimen- 
tally, ball valve prostheses have been inserted 
into the tricuspid position in a large number of 
calves. Some animals have received no specific 
treatment postoperatively, others have been 
given dextran and Coumadin, and in a third 
group the valves were coated with a solution of 
graphite-benzalkonium-heparin. Preliminary 



NATIONAL HEART INSTITUTE 



147 



observations indicate that anticoagulation is 
the most effective means for preventing 
thrombus formation with the valves in this po- 
sition. In other studies in dogs, however, in 
which the coagulation mechanisms differ from 
the calf, the graphite-benzalkonium-heparin 
coating seems quite effective. This species dif- 
ference will be resolved by continuing studies 
to determine the optimal plan of management 
for clinical application. 

The clinical and hemodynamic manifesta- 
tions of incomplete persistent A-V canal are 
now generally recognized but, because the mal- 
formation is relatively uncommon, there has 
been little information concerning the effects 
of operation on the circulatory abnormalities 
present preoperatively. The hemodynamic 
effects of operation were studied in 30 patients 
in whom operative treatment of this malforma- 
tion had been carried out. In all, the left-to- 
right shunt was immediately abolished, but re- 
curred in two patients in the late postoperative 
period. The operation was uniformly effective 
in reducing elevated pulmonary vascular re- 
sistance, and late studies demonstrated normal 
mitral and tricuspid valve function in all but 
two patients. In them, mitral incompetence was 
of principal importance preoperatively, indi- 
cating that prosthetic valve replacement may 
be necessary when the preoperative assess- 
ments indicate severe valvular regurgitation. 

Serum hepatitis remains an important com- 
plication of open heart surgery, and occurs in 
approximately 12 percent of patients operated 
upon in this Clinic. It has not been determined 
whether gamma globulin may prevent serum 
hepatitis, as it does infectious hepatitis, and 
the effectiveness of the agent was assessed in a 
blind study of 200 patients. Half were given 
gamma globulin before and 30 days after oper- 
ation, while the others received no specific pro- 
phylaxis. The incidence of hepatitis was simi- 
lar in the two groups. 

A previous report described an experimental 
study concerning the distribution of blood flow 
to the lungs after a subclavian-pulmonary ar- 
terial anastomosis. This indicated that a major 
proportion of the blood shunted from the sub- 
clavian artery passed to the lung on the side of 
the anastomosis, and that the shunt flow di- 
verted a major part of the right ventricular out- 



put to the lung on the side opposite the anasto- 
mosis. Recently, a new method for quantifica- 
tion of regional pulmonary blood flow in man 
has been developed. Macroaggregated human 
albumin, tagged with I^^^, may be injected 
either intravenously or into the aortic root, and 
the distribution of the particles in the lungs de- 
termined by external scanning. In cooperation 
with the Cardiology Branch, the technique is 
being applied in a systematic study of the pat- 
terns of pulmonary blood flow before and after 
operation in patients with various congenital 
malformations. Preliminary studies indicate 
that the observations made in normal dogs, 
after the Blalock operation, also apply in chil- 
dren, and that gross differences in the distribu- 
tion of shunted and nonshunted blood also oc- 
cur with patent ductus arteriosus. 

Idiopathic hypertrophic subaortic stenosis 
has become a well recognized clinical entity. 
Previous reports have described the studies in- 
dicating that in this disease outflow obstruc- 
tion is intensified when the volume of the left 
ventricle is reduced, and its contractile force 
increased. A number of reports from other la- 
boratories have stated that these mechanisms 
can cause outflow obstruction within the nor- 
mal left ventricle of the dog under conditions 
of hemorrhagic shock and/or the administra- 
tion of inotropic drugs. This hypothesis was re- 
examined, and particular attention was given 
to the methods utilized for measuring left ven- 
tricular pressure. It was conclusively demon- 
strated that the pressure gradient between the 
aorta and the left ventricle, which is often ob- 
served in normal dogs subjected to shock, is the 
result of artifact, and that there is no valid he- 
modynamic evidence that outflow obstruction 
can be induced in the normal left ventricle by 
any known intervention. The question as to 
whether factitious ventricular pressures can 
also be recorded in man is presently under in- 
vestigation. 

The normal patterns and volumes of arterial 
blood flow have been the subject of intensive 
study in this and other laboratories, but sur- 
prisingly little attention has been given to the 
flow patterns in the venous circulation. With 
specially designed intravascular flow trans- 
ducers, instantaneous blood flow was deter- 
mined in the venae cavae of dogs and related to 



148 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the pressure events in the right atrium and 
right ventricle. In normal animals, the flow 
pattern was phasic and least flow occurred at 
the peaks of atrial pressure. Similar relation- 
ships were observed in animals in which tricus- 
pid regurgitation, pulmonary stenosis, or var- 
ious arrhythmias were induced. Retrograde 
flo wof large magnitude was observed with 
those interventions which resulted in abnor- 
mally high peak atrial pressures. An exception- 
ally interesting abnormality of venous pressure 
and flow has been observed in both animals and 
man in the presence of ascites. The presence of 
severe ascites results in an elevation of the 
pressure in the inferior vena cava, while the 
right atrial pressure usually remains normal. 
In the dog, hemodynamic and angiographic 
studies indicate that acute ascites results in 
significant obstruction of inferior vena caval 
flow, probably caused by angulation of the cava 
at the diaphragm. In a limited number of pa- 
tients similar observations have been made, 
and in them it has also been observed that var- 
iations in body position profoundly affect the 
pressure gradient when it is present. In con- 
tinuing studies in both man and animals, the 
possibility that ascites may perpetuate itself by 
producing hepatic venous pressure elevation is 
to be investigated. 

It is well recognized that morphine is of 
great therapeutic value in the treatment of pa- 
tients with acute left ventricular failure and 
pulmonary edema, but surprisingly little is 
known of the mechanisms by which this drug 
alters cardiovascular performance. In normal 
dogs it was found that morphine resulted in an 
increase in myocardial contractile force of 
more than 50 percent, and also that ventricular 
function, as evaluated by function curves, im- 
proved strikingly after morphine injection. 
The mean rate of left ventricular ejection and 
left ventricular dp/dt showed similar improve- 
ment. In studies in which venous tone was 
measured, morphine resulted in the pooling of 
a significant fraction of the circulatory blood 
volume, indicating that it also has a depressor 
effect on the capacitance vessels. The effects of 
the drug will be further studied in animals in 
which acute left ventricular failure and pul- 
monary edema have been produced. 



General body hypothermia is often used in 
the treatment of patients with acute infections, 
neurologic disorders, or during operations in 
which a thoracotomy is not performed. In such 
circumstances the threat of ventricular fibrilla- 
tion is always present, and there has been re- 
newed interest in methods for protecting the hy- 
pothermic heart from this arrhythmia. The 
ventricular fibrillation threshold was studied in 
dogs rendered hypothermic with an extra-cor- 
poreal circuit, and alterations in the threshold 
were recorded when the arterial blood pH was 
normal and when it had been increased by the 
administration of THAM. When the pH was 
7.6 or higher the threshold increased by 50 per- 
cent or more, indicating that induced metabolic 
alkalosis may provide significant protection 
against ventricular fibrillation during hypoth- 
ermia. 

The effects of coupled and paired electrical 
stimulation on cardiac performance continue to 
be of interest in both the laboratory and recov- 
ery room. Recent studies have compared the 
effects of single and paired pacing in animals 
with complete heart block alone, and in animals 
with heart block complicated by cardiac fail- 
ure. Paired pacing dramatically improved the 
performance of the blocked failing heart, but 
was little better than single pacing in the ani- 
mals with heart block alone. The possible use- 
fulness of this method in the treatment of post- 
operative patients with myocardial failure is 
under continuing study in both the operating 
room and intensive care unit. 

In collaboration with Battelle Memorial In- 
stitute a new biologic adhesive was developed, 
consisting of a gelatin-resorcinol mixture po- 
lymerized with formalin. The material was not 
found to be suitable, as had been hoped, for the 
sutureless application of intracardiac prosthe- 
ses. In another experimental study, however, it 
was found quite effective as a method for con- 
trolling bleeding from cut surfaces of liver or 
kidney. The adhesive system is undergoing mod- 
ification so that only a single solution need be 
applied, and that better tissue bonds can be 
achieved in the presence of moisture or blood. 

Several clinico-pathologic investigations 
have resulted from the establishment of the 
pathology unit within the Clinic of Surgery. 



NATIONAL HEART INSTITUTE 



149 



Ten patients were studied in whom severe mi- 
tral regurgitation suddenly occurred as the re- 
sult of ruptured chordae tendineae. In contrast 
to patients with the usual form of rheumatic 
mitral regurgitation, these patients showed 
little left atrial enlargement, maintained sinus 
rhythm, and their symptoms, which appeared 
abruptly, usually led to death or necessitated 
operative treatment within a short time. In 
them, the mitral valves were normal except for 
the ruptured chordae, and in virtually all the 
etiology of the valvular malformation appeared 
to be bacterial endocarditis. In a review of 
more than 200 hearts of patients with valvular 
abnormalities, four were found to have severe 
aortic stenosis resulting from a congenitally 
malformed aortic valve which consisted of only 
a single cusp and a single commissure. Both 
operative and pathologic observations indicate 
that when such a congenitally stenotic valve is 
encountered, it is not amenable to commissuro- 
tomy since gross aortic regurgitation will cer- 
tainly result. Excision and prosthetic replace- 
ment of the unicommissural valve is the only 
means by which hemodynamic function can be 
restored. The carcinoid syndrome is generally 
well characterized, but the cardiac complica- 
tions continue to be of interest. Most recently it 
has been demonstrated that the endocardial 
fibrous lesions characteristic of the disease are 
often found within the wall of the right atrium 
as well as on the tricuspid valve, and this atrial 
fibrosis may prevent the chamber from expand- 
ing normally during right ventricular systole. 
As the consequence of this decreased com- 
pliance, right atrial pressures may be exces- 
sively elevated and lead to severe symptomology 
in patients with carcinoid heart disease and 
only trivial tricuspid regurgitation. 

GERONTOLOGY BRANCH 

Final plans have been completed for the de- 
sign of the Gerontology Research Building to 
be constructed at the Baltimore City Hospitals, 
Baltimore, Maryland. Members of the profes- 
sional staff have contributed considerable time 
and effort in working out the design of many 
special features of the building essential to the 
development of an effective research program 
on the phenomena of aging. 



Aging in the Human 

The series of repeated tests on the group of 
normal volunteers aged 20-96 years (called the 
Longitudinal Group) has been continued. Since 
the beginning of the program in 1958, 508 sub- 
jects have joined the group and have been test- 
ed once. In this group, 347 have been tested 
two times, 204 three times, 97 four times, and 
25 five times at intervals of 18 months. Twenty 
subjects in the sample have died but only 7 
have resigned from the study. 

All information obtained from the histories 
and physical examinations on each subject has 
been entered into the data retrieval system, 
and progress is being made on entering the 
backlog of numerical data. A major effort is 
being made to enter all data on punched cards 
and magnetic tape. Significant new additions to 
the testing program include the measurement 
of auditory and visual acuity, intra-ocular 
pressure by tonography, and the measurement 
of bone cortex thickness as an additional index 
of bone mass. 

Although a systematic analysis of the longi- 
tudinal aspects of the data must await the com- 
pletion of more testing sessions for each indi- 
vidual, certain aspects of the data already col- 
lected have been analyzed for age trends by the 
cross-sectional method. Results of these anal- 
yses (pulmonary function, glucose tolerance, 
EEG, frequency, learning ability) will be de- 
scribed in detail later in this report under the 
organ system involved. 

Physiological Basis of Behavior 

Further evidence shows that frequency of 
the electroencephalogram (EEG) is a critical 
factor both in understanding the timing of 
simple behavior and in interpreting age-asso- 
ciated differences in performance. Current 
findings indicate that involuntary reactions are 
inversely related to EEG frequency. In the 
same way, voluntary reactions were shown to 
be related to EEG frequency by our previous 
research. We have now found that latency of 
blocking of the EEG is positively correlated 
with EEG period (reciprocal of frequency). 
Blocking latency increases with age but here 
again it appears, as it did with voluntary re- 
sponse speed, that differences in EEG fre- 



150 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



quency which are associated with age may ac- 
count for this relationship. There is now sub- 
stantial evidence that frequency of the EEG is 
a critical factor in the timing of nervous sys- 
tem activity, and in the "slowing down" which 
is associated with behavior of older persons. 

Old subjects show a tendency to be less re- 
sponsive with respect to evocation of EEG 
blocking responses than young subjects. This 
result parallels another current finding that 
frequency of spontaneous galvanic skin 
reflexes (responses not associated with specific 
stimuli) is significantly lower in old persons 
than in young. Further tests of the generality 
of these specific findings are in progress. 

Frequency of spontaneous galvanic skin 
reflexes may serve as a physiological index of 
vigilance. Current research shows that subjects 
who perform a watch-keeping task exhibit 
fewer such spontaneous responses in an inter- 
val of time preceding a signal that is missed 
than in the period preceding a signal that is 
detected. 

Age Changes in Psychological Performance 

It has been hypothesized that interference 
from the presentation of other items of a learn- 
ing task is greater for the old person than for 
the young, and that this age-related susceptibil- 
ity to interference accounts for the large age 
differences found at fast learning paces in ear- 
lier studies. However, recent experimental evi- 
dence does not support interference as a psy- 
chological explanation for age deficits in verbal 
learning. One of the verbal-learning procedures 
used in the laboratory can be separated into 
three time intervals for each aspect of learn- 
ing: time to inspect the material, time to re- 
spond, and time between items. These time in- 
tervals were independently varied in a series of 
experiments. Previous research indicated that 
old subjects benefited more than the young by 
increasing the time permitted to respond. Cur- 
rent results suggest that old subjects do not 
benefit by increasing the time between items. 
These findings are not consistent with the in- 
terference hypothesis. 

On a logical problem-solving task requiring 
analysis and synthesis of information, a pre- 
vious finding from another laboratory indicat- 



ed that the performance of subjects above 60 
was inferior to the performance of young sub- 
jects. Some of the time intervals of the task 
were not under the subject's control. Similar 
problems were presented in this laboratory to 
men aged 28-81 with all time intervals con- 
trolled by the subject. The results were compat- 
ible with the earlier findings. Until the late 
50's no age differences were found, but after 
the age of 60 performance on a logical prob- 
lem-solving task declined even when no aspects 
of the task were paced. 

In perceptual studies, interference from 
judgments of interpolated stimuli has been 
shown to be greater for old men than for 
young. The measure of interference in time 
judgment was based on the increase in the 
number of errors in judging a set of short in- 
tervals before and after an interpolated set of 
longer intervals. To explore the nature of this 
type of interference, groups of old and young 
subjects were given, in addition to the interfer- 
ence task, another time judgment task which 
provided measures of accuracy, discriminabil- 
ity, and context effects. The new task required 
the subject to judge whether 220 visual stimuli 
were longer or shorter than one second. The 
first 110 stimuli were equally distributed 
around one second and the next 110 stimuli 
were predominantly longer than one second. 
Accuracy was measured for each subject by the 
difference between his subjective second (based 
on the first 110 judgments) and the clock sec- 
ond. Discriminability was measured by the var- 
iability of the first 110 judgments. A compari- 
son of the subjective second for the symmetri- 
cal set and for the set of predominantly longer 
intervals provided a measure of context effects 
due to the shift in the distribution of stimuli. 
Although no age differences were found for the 
measures of accuracy, discriminability, and 
context effects, the pattern of correlations with 
the measure of interference was diflferent for 
the two age groups. Interference was related to 
accuracy and discriminability for the old 
group, but for the young interference was re- 
lated to context effects. 

Pulmonary Physiology 

Spirometry was performed in 410 men who 
comprise the bulk of the longitudinal studies 



NATIONAL HEART INSTITUTE 



151 



population. These men were judged to be free 
of broncho-pulmonary and muscular disease by 
medical history, physical examination, chest x- 
ray and electrocardiography. Details of their 
smoking habits were obtained from a question- 
naire which was reviewed with them. For pur- 
poses of analysis, the subjects were divided 
into four groups : (1) 152 non-smokers, (2) 118 
current cigarette smokers, (3) 93 former ciga- 
rette smokers, and (4) 47 pipe and cigar 
smokers (past and present). It was found that 
there were no current cigarette smokers above 
age 69. For purposes of comparison, therefore, 
subclassifications of the other groups were 
made by eliminating subjects above age 69. 
The resulting subgroups were comparable with 
respect to age distribution. 

The evidence that cigarette smoking impairs 
spirometric function was twofold: (1) Covar- 
iance analysis indicated that the vital capacity 
(VC), forced expiratory volume at 0.5 seconds 
(FEV 0.5) , forced expiratory volume at 1.0 sec- 
ond (FEV 1.0), and maximum voluntary ven- 
tilation (MW) of the current cigarette 
smokers (N = 118) were significantly lower 
than those of a comparably constituted group 
of non-smokers (N = 122). (2) Multiple regres- 
sion analysis of the data of the current 
smokers indicated that, when adjustment was 
made for interrelated effects of age and 
height, the greater the consumption of ciga- 
rettes (measured as packages per day or pack- 
ages per day times years) the lower were the 
observed spirometric values. These regressions 
were significant for the same parameters as in 
the covariance analysis. 

Similar multiple regression analyses of the 
data of former cigarette smokers indicated 
that the greater the number of years smoked 
prior to cessation of smoking, the lower were 
the observed spirometric values. In this case, 
the regressions were significant for FEV 0.5, 
FEV 1.0, maximum mid-expiratory flow 
(MMF) and maximum expiratory flow rate 
(MEFR). In addition, when the number of 
years from cessation of smoking to testing was 
analyzed and adjustment was made for interre- 
lated effects of age, height and total consump- 
tion of cigarettes (as packages per day times 
years) , it was found that the greater the smok- 
ing-f ree interval, the greater were the observed 



spirometric measurements. This was signifi- 
cant for VC, FEV 0.5, FEV 1.0, and MEFR. 
Pipe and cigar smokers performed as well as 
non-smokers in all tests. 

It is concluded that, within the limitations of 
a retrospective, cross-sectional study, these 
findings constitute evidence that : (1) cigarette 
smoking impairs ventilatory function in sub- 
jects who appear healthy upon clinical evalua- 
tion, (2) this effect is related to the amount of 
cigarette consumption and (3) this effect is, in 
part, reversible upon cessation of smoking. 

More definitive evidence must await long 
term observation of subjects before and after 
cessation of smoking as is contemplated in the 
longitudinal studies in progress. 

Measurements of the uniformity of distribu- 
tion of air in the lungs have been summarized 
for 117 participants in the longitudinal studies 
program. The lung clearance index, a measure 
of this uniformity, showed that the distribu- 
tion of air in the lungs was less uniform in the 
old than in the young. An explanation of the 
poorer distribution of air found in the old par- 
ticipants was sought in the results of deep 
breathing tests. Nine young participants did 
not improve the uniformity of air distribution 
in their lungs when they breathed at three 
times their normal resting level while nine 
older participants improved the uniformity of 
distribution of air in their lungs significantly 
(as indicated by a reduced lung clearance in- 
dex) when they breathed at twice their normal 
resting levels. The mechanism of this effect is 
being investigated. 

Renal Physiology 

The effects of the ingestion of a number of 
substances on electrolyte excretion in old and 
young subjects was determined. The ingestion 
of ethanol, glucose, galactose and casein caused 
marked increases in the urinary excretion of 
calcium and magnesium while potassium excre- 
tion fell in young subjects. No consistent 
changes were observed in urinary sodium ex- 
cretion. Ingestion of xylose and fat failed to 
produce similar changes. The changes in dival- 
ent cation excretion were independent of var- 
iations in the glomerular filtration rate, urine 
pH, urine flow, urine total solute and carbohy- 



152 



ANNUAL KEVIEW OF INTRAMURAL RESEARCH 



drate excretion, urine organic (lactate, pyru- 
vate, urate, total organic acid) or inorganic 
(phosphate) anion excretion, and levels of se- 
rum calcium, magnesium and carbohydrate. The 
increase in urinary divalent cation excretion is 
a non-specific effect observed following the 
ingestion of nutrients capable of being readily 
metabolized in the kidney. 

Most of the old subjects failed to show these 
increases in divalent cation excretion following 
oral ingestion of 100 grams of glucose. The 
older subjects usually had sharp falls in uri- 
nary sodium excretion which obscured the in- 
creases in divalent cation excretion observed in 
the young. Periods of fasting of 36 hours 
tended to reproduce the pattern seen in the old 
subjects in young subjects. The mechanism of 
this response and the basis for the age differ- 
ences are being further explored. 

A study has been initiated to compare the 
ability of young and old subjects to clear an 
acute acid load through the kidneys. Acute oral 
acid loads, in the form of ammonium chloride 
gelatin capsules (0.1 gram per Kg. body 
weight) have been given to adult male volun- 
teers from 2 to 85 years of age. In all age cat- 
egories, initial serum pH, pCOa and total CO2 
were normal. An approximately equal fall in 
total CO2 content of blood serum was observed 
in all age groups following the acid load. By 
the end of eight hours the younger subjects had 
restored their serum total CO2 levels to normal 
while the older subjects still had markedly re- 
duced levels of serum total CO2. All subjects 
lowered urine pH to similar levels. The rate of 
ammonia excretion increased markedly in the 
young subjects but remained low in the old. 
These preliminary results indicate that aging 
is accompanied by a reduction in a metabolic 
function of the kidney. 

Experiments were conducted with dogs to 
study the mechanism of action of ethacrynic 
acid on the kidney. Stop flow studies showed an 
increase in distal minimum sodium concentra- 
tion and examination of medullary tissue 
showed a decreased concentration of sodium. 
These changes, along with studies of concen- 
trating and diluting ability, suggest inhibition 
of sodium transport in the ascending loop of 
Henle. 



Endocrinology 

Research on hypertensive disease has long 
been hampered by lack of chemical methods for 
the determination of renin and angiotensin. 
Heretofore all analytical techniques depended 
on bioassays, most of which are of questionable 
specificity and precision. 

A physico-chemical method for the measure- 
ment of renin and angiotensin in the blood is 
being developed. The method is based on the 
double isotope derivative assay which has been 
for steroid determinations. Dinitroflurobenzene 
(DNFB) has been found an effective reagent 
for reaction with angiotensin. The procedure 
is to react the unknown amount of angiotensin 
with DNFB-H^ following the addition of 
C"DNP angiotensin and unlabeled DNP 
angiotensin as indicator and carrier re- 
spectively. The DNP angiotensin is precipitat- 
ed, washed, extracted and chromatographed on 
a thin layer plate. The yellow DNP angiotensin 
spot is eluted and counted for tritium (a meas- 
ure of the original angiotensin content), and 
C" (a measure of the overall recovery of 
DNP derivative which permits correction for 
the tritium losses). As little as 0.1x10"^ gram 
of the peptide has been measured. The sensi- 
tivity of the method has been greatly enhanced 
by the development of DNFB-H= reagent with 
a specific activity that is 22 times greater than 
the reagent previously available. Work is now 
progressing on reducing the blank values 
which will make it possible to determine 
amounts of the peptide in amounts of 
0.1 X 10-'' grams. 

The importance of this project is both theo- 
retical and practical. If successful, this method 
would be the first isotope derivative assay to be 
developed for a small polypeptide hormone and 
would lead to the development of other assays 
of similar important peptides. A practical ren- 
in assay, on the other hand, would be a major 
step in the everyday hospital differential diag- 
nosis of hypertensive disease, since it would al- 
most certainly permit recognition of unilateral 
(ischemic) reno-vascular hypertensives. The 
problem has, therefore, a place in the attack on 
hypertension, a major age-related disease com- 
plex. In addition, renin and angiotensin are in- 
timately related to the secretion of aldosterone, 



NATIONAL HEART INSTITUTE 



153 



and adequate methodology is badly needed in 
this area of physiology and patho-physiology. 
An additional aspect of the program is a study 
of the interrelations between angiotensin, re- 
nin and aldosterone in normal old persons. 

Metabolism 

Studies of carbohydrate metabolism in the 
Longitudinal Group are yielding data for a 
cross-sectional analysis of results. The objec- 
tives of the study are: (1) to describe results of 
the commonly used tests for diabetes mellitus 
in a carefully studied group of men over the 
entire adult age span, (2) to investigate the 
causes of the decline in glucose tolerance with 
age, and (3) to discover which procedures will 
predict best the future development of overt 
clinical diabetes in patients who will be fol- 
lowed for a long time. 

Use of the intravenous glucose tolerance test 
(IVGTT) in clinical medicine has been inhibit- 
ed by recommendations for increasingly com- 
plex methods of analysis of the curve of glu- 
cose concentration following rapid glucose 
administration. By computer techniques we 
have shown that performance on the test can be 
judged as well simply by noting the glucose 
concentration at 60 or 80 minutes following in- 
jection as by any of a variety of complicated 
mathematical techniques as judged by high 
correlations between results obtained by differ- 
ent methods of summarizing the data. Perform- 
ance judged by the simple technique corre- 
lates as highly with age (r = 0.60) as do the 
complex techniques (r= 0.58-0.62). 

Serum insulin levels by radioisotopic double 
immuno-assay technique in the IVGTT show 
(1) initial average values of 10±0.7 (S.E.) /jiU 
per ml, (2) peak concentration at 10 minutes 
of 40±3.4, and (3) slow decline thereafter to 
23±1.9 at 80 minutes. There was no signifi- 
cant correlation between age and plasma insu- 
lin concentration at any time during the test 
despite the markedly slower rate of decline of 
blood glucose concentration with increasing 
age. These results imply that the age defect is 
not secondary to inadequate insulin production, 
but that decreased sensitivity to insulin might 
play a role. 



A series of oral glucose tolerance tests in the 
Longitudinal Group shows much greater varia- 
bility in response than with the intravenous 
test. The necessity of providing normal stand- 
ards which take into account the variable of 
subject age is shown by the fact that in the 
first 24 subjects over age 60 with no personal 
or family history of diabetes, 13 (58%) had 2 
hour glucose concentrations greater than 120 
mg per 100 ml, the commonly accepted upper 
limit of normal. Serum insulin responses in 
these subjects are also being examined. 

The first series of cortisone glucose tolerance 
tests (CGTT) have been completed. The test 
was used because of the exaggerated response 
of diabetics to steroid administration. Thus, if 
the factors causing deterioration in glucose tol- 
erance that accompanies aging were physiolog- 
ically distinct from those factors associated 
with diabetes, a divergent response to cortisone 
might have occurred. In fact, older subjects 
showed as accentuated response to cortisone as 
do diabetics. 

Serum insulin responses to the CGTT show 
that cortisone increases basal insulin concen- 
trations from 10 to 16/xU/ml. After oral glu- 
cose administration insulin levels increased 
progressively to a peak mean value at 80 min- 
utes of 126±8.7 (S.E.) in 60 normal subjects. 
During this rising phase there was no correla- 
tion of insulin levels with age. Mean values at 
100 and 120 minutes remained 123 and 
126/iU/ml, but this apparent plateau is 
caused by the fact that older subjects in the 
group tend to have a further increase in insulin 
concentration while younger subjects tended to 
have a falling concentration. Therefore, at 120 
minutes there was a positive correlation with 
age; insulin levels increase by 11 /tU.ml per dec- 
ade of adult life (p<.05). These results con- 
firm those of the IVGTT in that the decline in 
tolerance cannot be attributed to inadequate in- 
sulin response to hyperglycemia. 

A series of nomograms to aid in the inter- 
pretation of the clinical tests for diabetes have 
been prepared. These have been constructed 
for the IVGTT and the CGTT. They permit rap- 
id determination of the rank of an individual 
in comparison to his age cohorts. The nomo- 
grams consist of 3 linear scales: age, glucose 
concentration (at 80 minutes in the IVGTT, at 



154 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



120 minutes in the CGTT) , and a probit scale. 
A straight edge connecting appropriate points 
on the first two scales intersects the third scale 
at the percentile rank. 

Biology of Aging 

Behavioral Changes tvith Age 

Recent gerontological theory has attempted 
to explain age differences in motivation on the 
basis of physiological factors and in terms of 
differences in response to various environmen- 
tal factors. A general factor termed "rigidity" 
has also been proposed as an explanatory con- 
struct. The program of this section is concerned 
with (a) determining basic behavioral age dif- 
ferences, and (b) finding environmental fac- 
tors which reduce age differences. The use of 
lower organisms such as the rat is necessary 
for such a program. 

It was previously reported that naive adoles- 
cent rats (2 mo. old) and naive senescent rats 
(24 mo. old) obtained very low scores of explo- 
ration in an open field and remained within a 
dark hiding area, in contrast to very young (1 
mo. old) or young adult (3 mo. old) rats who 
obtained high scores for exploration. During 
the past year it was found that fearfulness of 
adolescent rats could be lowered with an attend- 
ant increase in exploration by means of (1) 
systematic gentling or (2) testing during the 
dark portion of the dark-light cycle. However, 
the scores of senescent rats were not changed 
by these environment variables. These data in- 
dicate that the fearfulness of adolescent rats 
may be decreased by a variety of environmen- 
tal variables, while the behavior of senescent 
rats is more rigid, in the sense that variables 
which reduce fear in young rats do not induce 
behavioral changes in senescent rats. 

Senescent nondeprived rats were found to 
make more responses to obtain a palatable food 
reward than did young rats. The senescent rats 
would be expected to persist in responses 
which were not "need" reducing if they were 
more rigid than the young rats. Senescent rats 
were shown to retain the habit of responding 
for reward, while younger rats showed less 
retention. 

Differences in performance between de- 
prived young and senescent rats working for a 



food reward (one response results in one re- 
ward) have not been observed. Also, tests of 
retention indicate that rates of response are 
similar for old and young rats over the series of 
trials given. However, if the senescent rats 
were more rigid than the young rats, an inter- 
posed stimulus (in this case, a stimulus corre- 
lated with food reward) should result in a 
more permanent change in the behavior of the 
senescent than the young group. Preliminary 
experiments have shown that the introduction 
of such a stimulus during retention tests re- 
sults in a more permanent change in response 
patterns for the senescent than young rats, 
while young rats switch back to a formerly re- 
warded response on later trials. 

If physical stamina decreases with increas- 
ing age, an increase in the amount of effort to 
obtain a food reward should result in increas- 
ingly large performance differences between 
young and senescent subjects. An experiment 
has been completed in which the number of re- 
sponses required to obtain a reward was pro- 
gressively increased. Performance differences 
between food deprived young and senescent 
rats became increasingly greater as the 
number of responses required for the reward 
increased. 

Physiological Systems — Heart 

Both the isolated heart-lung preparation and 
the isolated perfused heart from the rat have 
been used to determine the functional capacity 
of the myocardium, expressed as an index of 
left ventricular work (LVWI) in gm M/lOO 
gm dry heart weight. There was a large differ- 
ence between the work capacity of hearts from 
young adult (12 mo. old) rats and senescent 
animals (24 mo. old) . The index for heart-lung 
preparations from 12 month old animals was 
163 ±13 and for 24 month old animals it was 
30±4. The performance scores for the isolated 
perfused heart were 68±10 and 9±3 for 12 
month old and 24 month old animals, respec- 
tively. The work output of fatigued hearts could 
be significantly restored by adding corticoster- 
one at a concentration of 15 fj.g% to the blood 
perfusing the heart. Histological examination 
of the heart at the end of the experiment failed 
to show morphological changes sufficient to ac- 



NATIONAL HEART INSTITUTE 



155 



count for the large differences in performance 
between the young and old hearts. 

Connective Tissue 

In spite of many efforts to determine the 
effect of age on the structure of collagen, much 
of the evidence on the formation of cross-link- 
ages has been contradictory. Some of these 
contradictions have been resolved by the fol- 
lowing experiments with rat tail tendon colla- 
gen from 1, 3, 12 and 24 month old rats. From 
ultracentrifugation studies the relative quanti- 
ties of a-collagen (no cross-links) and ^S-colla- 
gen (consisting of units having two a-strands 
covalently cross-linked) have been determined 
in the acid soluble portion of the collagen. The 
fraction of ;S-collagen increases greatly be- 
tween 1 and 3 months, but relatively little 
change occurs thereafter. From solubilization 
studies it is apparent that both the rate of solu- 
bilization and the amount of collagen solubil- 
ized decreases greatly with age. Thus cross- 
linking in soluble collagen levels off after 
3 months, but cross-linking beyond the 2- 
stranded stage, represented by insoluble 
forms, appears to continue to increase with 
age. 

These results appear to rule out the simple 
picture that collagen from all age groups con- 
sists of varying amounts of the same kind of 
soluble and the same kind of insoluble collagen, 
the latter increasing due to aging as new colla- 
gen is laid down over it. Instead, the collagen 
that is soluble becomes increasingly inacces- 
sible to the solvent, and presumably is trapped 
in a network of massively cross-linking, insol- 
uble material. The levelling after 3 months in 
the concentration of /8-collagen in the soluble 
component may indicate that jS-coUagen has a 
greater tendency in older animals to cross-link 
further, resulting in the formation of more of 
the insoluble material. 

Nutrition and Aging 

Biochemical parameters have frequently 
been used to formulate hjTpotheses for the 
manner in which dietary restriction prolongs 
life span. Since it is well known that animals 
offered a restricted amount of food consume 
the entire ration within a short period of time 



whereas food is available to ad lib fed controls 
for 24 hours per day, it seemed necessary to 
determine whether differences in the interval 
of time between feeding and sacrifice account- 
ed for the previously reported differences in 
enzymatic activities between normal and 
restricted rats. In addition to the previously 
employed measurements of hepatic succinoxi- 
dase and renal alkaline phosphatase activities, 
protein synthesis was estimated by the incor- 
poration of C"-glycine into proteins of liver 
slices. Reduced food intake resulted in the ex- 
pected increments in the two enzymatic activi- 
ties and also in a decrement in C"-glycine in- 
corporation. Results obtained when the fasting 
interval was varied indicated that the biochem- 
ical alterations associated with dietary restric- 
tion were not due to a displacement in the cyclic 
variation in metabolic activities as a result of 
the time of feeding. 

In studies to determine the mechanisms re- 
sponsible for the influence of various environ- 
mental conditions on the life span of rotifers, 
animals maintained at different temperatures 
were subjected to dietary restriction. The mean 
life span was shortened by increased tempera- 
ture and was lengthened by dietary restriction 
at temperatures of 25°C to 35°C. However, the 
interval of time between the cessation of egg 
production and death was decreased by in- 
creased temperature but was unaffected by nu- 
trition. Thus, these two variables had preferen- 
tial effects on different physiological stages in 
the life cycle of these animals. Therefore, it 
seems that alterations in life span by these en- 
vironmental factors is not brought about by 
the same biological mechanism. 

Earlier experimental work showed that cul- 
tures of a streptomycin-bleached Euglena gra- 
cilis remained completely viable up to 12-14 
days in the absence of an external carbon 
source. The cells apparently survive through 
the formation of cytolysomes in their cyto- 
plasm. This process of autodigestion provides a 
source of carbon to the cells. In the past year, 
it has been observed that hydrolytic enzyme ac- 
tivity as exemplified by acid phosphatase, ca- 
thepsin and esterase, increases during carbon 
deprivation. Such enzyme activity correlates 
with the process of autodigestion. Further, 
there appears to be a time sequence in the hy- 



156 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



drolytic enzyme activity with esterase, the first 
to appear. It is not known yet whether the 
enzymes are actually synthesized during carbon 
deprivation or whether an inhibitor (repressor) 
of their activity is removed at this time. Under 
electron microscopy, carried out in collabora- 
tion with Dr. D. Brandes of the Baltimore City 
Hospitals, at least one of these enzymes (i.e., 
acid phosphatase) is seen to localize mainly in 
the Golgi apparatus and in the cytolysomes. 

This observation led to an attempt to isolate 
the cytolysomes. By means of a glass bead dis- 
ruption technique originally developed in this 
laboratory for the isolation of mitochondria 
from "normal" non-starved Euglena, cytoly- 
somes have been isolated in an intact state 
from carbon-starved Euglena. This marks the 
first isolation of such particles from any cell 
type. Acid phosphatase activity was preserved 
within the cytolysomes during fractionation 
and histochemical manipulations for electron 
microscopy. Further, the enzyme was distribut- 
ed identically as was found in intact cells. The 
isolation of cytolysomes will allow a study of 
their biochemical and morphological properties 
and, hopefully, will provide an insight into the 
mechanism of autodigestion by the cells. 

Earlier studies on the effect of actinomycin 
D on carbon-starved Euglena led to the conclu- 
sion that those ribosomes not digested away 
during starvation were "stabilized", i.e., re- 
mained "programmed" for protein synthesis 
and therefore could synthesize protein if amino 
acids in proper form were available. Therefore, 
if carbon-starved cells were fractionated and 
the appropriate fractions combined and amino 
acids provided, cell-free protein synthesis 
should take place in the test tube. In the last 
year this was accomplished. Fractions from 
carbon-starved Euglena show protein synthesis 
at rates similar to the control non-starved cells. 
It will be of interest to fractionate further the 
protein synthetic system in order to determine 
the nature of the components which persist 
over long periods of time. Another problem to 
be investigated is whether or not the protein 
Rvtithesis system becomes oriented in the direc- 
tion of hydrolytic enzyme production during 
carbon deprivation in Euglenu. 

The formation of chloroplasts in Euglena 
has been shown by other investigators to be ac- 



companied by large scale RNA and protein 
synthesis. It was felt that study of chloroplast 
formation in Euglena would be useful in eluci- 
dating cell control mechanisms involving the 
formation of specific cytoplasmic structures. It 
is of further interest that the chlorophyll con- 
tent of Euglena chloroplasts reaches a maxi- 
mum after the cells have entered the post-mitot- 
ic or stationary phase of their growth cycle. 
Chlorophyll synthesis in these cells is sensitive 
to such inhibitors as streptomycin and fluorou- 
racil as well as high temperature. In the past 
year it has been found that streptomycin is 
most inhibitory under conditions that allow cell 
division. Further, Euglena is permanently 
bleached at high temperature after about 9 cell 
divisions in a culture. These results suggest the 
prevention of replication and subsequent "di- 
luting out" with time of some factor essential 
for the development of a complete chloroplast. 
Fluorouracil also inhibits "greening" in Eu- 
glena. 

"Error" Theory of Cellular Aging 

One of the current theories of aging pro- 
poses that with senescence, alterations occur in 
the structure of the DNA molecule. This error 
is transmitted to messenger RNA and ultimate- 
ly to newly synthesized enzymes. These de- 
fective enzymes may be inactive and therefore 
an accumulation of substrates within the cell 
may take place. This may stimulate an increase 
in RNA and protein turnover to compensate 
for the defective enzymes. If the production of 
inactive enzymes proceeds to the point that in- 
creased synthesis cannot compensate for the 
error, death of the cell and ultimately of the 
organism occurs. During the past year studies 
were initiated to test this hypothesis. Two ap- 
proaches were used. The first was to examine 
various tissues of rats for age-associated 
changes which would be predicted to result 
from the structural errors proposed in this 
theory. The second method was to determine 
whether the experimental introduction of er- 
rors into protein molecules would also result in 
the same biochemical alterations. Data which 
have been obtained on a small number of adult 
and old rats indicated age-dependent increases 
in the catabolism of nucleic acid and protein as 



NATIONAL HKAET INSTITUTE 



157 



estimated by the activities of hepatic ribonu- 
clease and cathepsin respectively. These results 
are consistent with the error theory. However, 
in these same animals age-associated decre- 
ments rather than the predicted increments 
were observed in the incorporation of C"-uri- 
dine into liver RNA, in incorporation of 
C"-leucine into proteins of liver microsomes 
and in the C "-leucine uptake by liver slices in 
the absence of added essential and non-essen- 
tial amino acids. Furthermore, no qualitative 
changes with age were observed in the pattern 
of lactic dehydrogenase isoenzymes, which 
represent structurally different forms of the 
enzyme. Thus, these latter techniques failed to 
yield data which would completely suppoi't the 
error theory of aging. 

The second approach to testing the hypoth- 
esis was carried out by feeding animals diets 
which contained an analogue of an essential 
amino acid in order to introduce errors into en- 
zymes. Several preliminary studies have been 
carried out in which female rats were fed diets 
which contained various amounts of ethionine, 
an analogue of the essential amino acid, me- 
thionine. The results have indicated that the 
animals failed to survive when fed a diet which 
contained 1/2% ethionine. Feeding levels of 1/ 
8% or 1/4% of the analogue to adult rats 
(10-12 mo. old) resulted in initial losses in 
body weight which subsequently became stable 
and had no apparent toxic effect on the livers. 
The result of biochemical analyses of the 
tissues were consistent with the proposed 
hypothesis, i.e., increases in the C"-glycine 
incorporation and in the activities of cathepsin 
and ribonuclease in liver tissue were found in 
the ethionine-fed animals. However, in a subse- 
quent study carried out on younger (3-4 month 
old) rats fed 1/8% ethionine, not all of the 
metabolic patterns compatible with the theory 
were apparent in the experimental group. De- 
velopment of fatty livers was indicated by the 
gross appearance of the tissue as well as by an 
increased liver weight to body weight ratio in 
the ethionine-fed group. It was concluded that 
a higher food intake, and hence higher ethio- 
nine ingestion, per unit of body weight in young 
than in adult animals accounted for the discrep- 
ancy between the two studies. Therefore, at 



present final proof that age-associated changes 
are due to errors is not available. 

Age Pigment 

Studies on the properties of lipofuscin age 
pigments have been continued, and it has beer- 
shown that the so-called lysosomal enzymes 
(esterase, cathepsin) are not associated with 
the most highly purified age pigment prepara- 
tions of human liver. It therefore appears 
either that the age pigments in this tissue are 
not of lysosomal origin or have lost enzyme ac- 
tivity. On the other hand, isolated lipofuscin 
from human and bovine heart does show the 
presence of esterase and catheptic activity, 
although many individual particles appear to 
be inactive. 

Mechanisms of Cell Death 

During the year, a considerable number of 
changes occurring at the histological level 
during the process of cell death and aging have 
been identified. In collaboration with Dr. 
Wilder's group at Baltimore City Hospitals, ex- 
perimental infarcts were produced in a group 
of 50 dogs. These animals were sacrificed at in- 
tervals from 1/2 hour to 20 days after tying off 
a branch of the anterior coronary artery. The 
infarcted zone, plus control tissue, was re- 
moved and a portion fixed and another portion 
frozen immediately, following which an exten- 
sive battery of histochemical and histoenzymo- 
logical tests were applied. The most striking 
early changes occurring in cells during the proc- 
ess of cell death included the disappearance of 
choline-containing lipids, the accumulation of 
neutral lipids and phospholipids, the depletion 
of glycogen and an increase in several enzyme 
activities, notably succinoxidase, esterase, and 
phosphatase in association with mitochondria. 
At later stages of infarction, a PAS positive 
material appeared (not glycogen) and there 
was a striking increase in leucine aminopepti- 
dase, and the disappearance of phospholipids. 

These findings have furnished the basis for a 
biochemical study of events accompanying cell 
death. These studies have been undertaken in 
collaboration with Dr. Prem Batra of the Ag- 
ing Research Laboratory, Veterans Adminis- 
tration Hospital, Baltimore, and consist of 



158 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



measurements of adenine nucleotide levels and 
phosphorylative efficiencies in mitochondrial 
preparations isolated from damaged or dying 
tissues. This work has resulted in the develop- 
ment of a new and specific assay procedure for 
oxidative phosphorylation, which utilizes a 
modification of the Strehler-Totter firefly 
luminescense assay. This procedure permits the 
measurement of phosphorylative rates and 
P/0 ratios within one to two minutes on indi- 
vidual preparations. 

Changes during the aging of the firefly lan- 
tern (Photinus pyralis) were studied by the 
same battery of the histochemical tests applied 
to the cell death studies. Age-related decreases 
were noted in the levels of succinoxidase, 
ATPase and ADPase. Drastic decreases were 
observed in stored glycogen in both the reflec- 
tor layer and the photogenic layer, which were 
accompanied by marked declines in RNA con- 
centration, considerable decreases in phos- 
pholipids by the luxol and Sudan methods, and 
declines in the concentration of basic proteins, 
and in tryptophane, sulfahydra, and R groups. 
These changes probably reflect, in part, intrin- 
sic age changes and, in part, are a result of the 
limited feeding habits of the adult Photinus py- 
ralis. 

Hibernation. — In studies of hibernation, it 
has been shown that there is a considerable 
change in the content of "apparent glucose" in 
hamster erythrocytes after the animals have 
been cold-exposed for several weeks. About 30 
to 45 times more glucose oxidase reacting sub- 
stance is present in the controls and short-term 
cold exposed animals than in cold-exposed ani- 
mals just prior to hibernation and in hiberna- 
tors. The changes in adenine nucleotides and 
phosphorylated glycolj^ic intermediates ob- 
served were less substantial when hibernators 
were compared to controls. Both reduced body 
temperatures during hibernation and active 
metabolic controls seem responsible for the 
changes in glycolytic pools. 

In attempts to localize the thermal control 
center of the brains of hamsters by histochemi- 
cal means paired hypothalami of hibernating 
and non-hibernating animals were sectioned si- 
multaneously and serially. These sections were 
then exposed to a variety of histoenzymological 
tests. No differences were noted in enzyme con- 



centrations between the two groups. However, 
a series of highly specific enzyme localizations 
was observed in several of the hypothalamic 
nuclei. These nuclei included the habenular and 
interpeduncular and associated tracts which 
stained much more intensely for esterase than 
any other neural structure, and the suprachias- 
matic nucleus which stained intensely and spe- 
cifically for alkaline phosphatase. Studies of 
other species indicate that this enzyme distri- 
bution pattern is uncommon and not character- 
istic of all species capable of hibernation, but 
rather varies widely from species to species. 
The significance of these striking local concen- 
trations of enzymatic activity cannot at pres- 
ent be evaluated since the function of these 
structures has not yet been determined. 

Basic Biology 

In many instances, investigations of the role 
of cellular processes in longevity and aging are 
hampered because of inadequate knowledge 
about the basic mechanisms involved. Hence, 
part of the research program of the Gerontol- 
ogy Branch is devoted to basic research in cel- 
lular biochemistry and molecular biology. 

Intermediary Metabolism 

Previous work had demonstrated that the 
oxidation of acetate-2-C" to C^Oa occurred 
more rapidly in senescent rats than in adult 
rats. In order to explain the molecular basis for 
the above difference, a detailed knowledge of 
the mitochondrial reactions is required since 
they are concerned with the terminal oxidation 
of acetate. 

The studies on the mechanism of oxidative 
phosphorylation in beef heart mitochondria 
have been continued. The work has centered on 
the first site of phosphorylation; viz., that oc- 
curring in the segment of the respiratory chain 
between NADH and cytochrome b with some 
probing of phosphorylating reactions at other 
sites. The reactions at the first phosphorylation 
site may be formally illustrated as in Fig. 1. 
The goal of the research is to isolate each of 
the unknown components in order to reconsti- 
tute the reaction with purified fractions. The 
purification of coupling enzyme 1 (CE 1) was 
reported in previous years. An assay for a sec- 



NATIONAL HEART INSTITUTE 



159 



NADH > Flavoprotein 

\ 



1 



Coenzyme Q 



I 



I Coupling enzyme 1 



ATP 



Fig. 1 



ond coupling- factor has been devised and its 
purification is in progress. It has been found 
that submitochondrial particles, depleted of en- 
dogenous coupling factors by treatment with 
urea and supplemented with a saturation level 
of CE-1, are still deficient in other factors. 
Under these conditions, additional stimulation 
of ATP-dependent NAD reduction by succinate 
(reversed oxidative phosphorylation) was ob- 
tained with a crude soluble extract of mito- 
chondria. The new factor is heat-labile, 
nondialyzable and can be fractionated by pre- 
cipitation with ammonium sulfate and adsorp- 
tion on DEAE-cellulose. The preparation of the 
factor also catalyzes an exchange of ATP with 
^^Pi. However, since the reaction is insensitive 
to oligomycin, the relationship of this activity 
to oxidative phosphorylation is not established. 

Evidence has been obtained indicating that 
the flavoprotein shown in Fig. 1 may be identi- 
cal with the NADH-CoQ reductase whose 
extraction from mitochondria and partial purifi- 
cation was reported earlier. The CoQe reduc- 
tase activity and its sensitivity to rotenone ap- 
pear to be unique to this particular enzyme 
since preparations of NADH dehydrogenase 
made by procedures devised in other laborato- 
ries gave no activity with CoQs. It appears that 
the activity with CoQe requires preservation of 
a labile site on the flavoprotein or an addition- 
al tightly bound component. 

Further evidence that the non-heme iron in 
the NADH-CoQ reductase is not catalytically 
active in the oxidation has been obtained using 
the EPR spectrometer. In collaboration with 
Dr. H. Beinert, it was found that the enzyme 
had an EPR signal at g=4.3 which disap- 
peared on addition of excess NADH at a very 
slow rate compared to the turnover rate of the 
.flavin. The high-spin ferric iron responsible for 



the signal apparently does not participate in 
the electron transfer. This confirms previous 
data obtained by binding the iron with o-phen- 
anthroline. 

Recent work has demonstrated that the 
preparation of NADH-CoQ reductase actually 
consists of two flavoproteins, both of which 
have essentially the same activity with mena- 
dione but one is twice as active as the other 
with CoQe. Further characterization of the two 
fractions is in progress. 

It has been established in other laboratories 
that non-phosphorylated high-energy interme- 
diates (analogous to '-'i in Fig. 1) generated at 
the oxidation site between cytochrome c and 
oxygen (Site 3) may be transferred to Site 1 
and utilized to drive the reduction of NAD by 
succinate. Considerable effort was directed to- 
wards separation of soluble factors associated 
with the reaction since the activity is indepen- 
dent of the terminal CE-1 reaction. It was 
found that the system, present in submitochon- 
drial particles, was relatively stable and affect- 
ed but little on exposure to 2 M urea, pH 11, 
and trypsin. Addition of several types of pro- 
tein and non-protein extracts and lipids did not 
stimulate the activity in the treated particles. 

An assay specific for the phosphorylation 
site between cytochrome b and cytochrome c 
(Site 2) has been devised. It consists of reduc- 
ing endogenous cytochrome c with ascorbate in 
the presence of a suitable dye and then driving 
the electrons to reduce cytochrome b at the ex- 
pense of energy from added ATP. Specific as- 
says are now available for each site of oxida- 
tive phosphorylation. 

The energy-dependent cytochrome b reduc- 
tion had the expected sensitivity to uncoupling 
agents and electron transfer inhibitors. 

Molecular Biology — Properties of DNA and 
RNA 

The ability of copper (II) ions to unwind 
completely and, under different solution condi- 
tions to rewind the two strands of DNA com- 
pletely, has been proved beyond reasonable 
doubt, and the mechanism of the denaturation 
process has been established as consisting of 
the interpolation of relatively few copper ions 
between complementary bases and the subse- 



160 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



quent initiation of a gradual unwinding of the 
remainder of the double helix. The equilibria 
involved in these reactions are believed to con- 
stitute a model of the unwinding and rewind- 
ing of DNA that occurs during its replication 
in the cell. Copper (II) ions can unwind and 
rewind the poly (A + U) molecule in the same 
way as DNA. This reaction is therefore also a 
model for the attachment and subsequent 
detachment of messenger UNA from DNA. It 
should be emphasized that these reactions are 
models and not biochemical mechanisms since 
no participation of copper in the biological proc- 
esses has been demonstrated. 

The degradation of RNA by cleavage of the 
phosphodiester linkages with zinc proved to be 
non-specific, contrary to the anticipation from 
the previous finding that the homopolyribonu- 
cleotides are degraded at different rates. The 
reaction is therefore not useful in sequence de- 
terminations, although its possible importance 



in the biological degradation of ENA should be 
investigated. 

The biological functions of the nucleic acids 
make it necessary that the following four chem- 
ical reactions take place: (1) the formation 
of phosphodiester linkages, (2) the destruction 
of these linkages, (3) the ordering of the mole- 
cules into helical structures, and (4) the un- 
winding of these helical structures. It has now 
been shown that all but the first of these reac- 
tions can be brought about by metal ions. Reac- 
tions (2) amd (3) are mitigated by metal ions 
binding to the phosphate moities, whereas 
reaction (4) is initiated by metals binding to 
the bases. Conditions have been found under 
which some metal ions bind to the bases while 
other metal ions simultaneously bind to phos- 
phate. In view of these findings, it is reason- 
able to suppose that metal ions can be useful in 
the regulation of the biological activities of the 
nucleic acids. 



NATIONAL INSTITUTE OF ARTHRITIS AND 
METABOLIC DISEASES 



INTRODUCTION 

The past year has seen several changes in 
the NIAMD, the major one resulting from the 
untimely death of Dr. Joseph J. Bunim who 
was both Clinical Director and Chief of the 
Arthritis and Rheumatism Branch. Dr. Bunim 
had served as Clinical Director of NIAMD 
since the first days of Clinical Investigations at 
NIH. Under his guidance research in arthritis 
and rheumatic diseases has prospered both at a 
clinical and basic level. His death removed a 
backbone of the Institute. We have recently 
been fortunate in obtaining Dr. Robert S. Gor- 
don, Jr. as Clinical Director. Dr. Gordon has 
been with the NIH for 14 years and spent 
three of them as Chief of Clinical Research of 
the Pakistan-SEATO Cholera Research Labo- 
ratory. 

Honors have come to many of the staff. The 
Mead Johnson award to Dr. John Bieri for his 
work in nutrition, a well deserved honor, may 
be noted. Dr. Bruce Ames' delineation of the 
biochemistry and genetics of the pathway for 
the biosynthesis of histidine has resulted in 
several honors: The Eli Lilly award of the 
American Chemical Society and the Biological 
Sciences Achievement Award of the Washing- 
ton Academy of Sciences. Dr. Robert Schimke's 
studies on the control of enzyme levels resulted 
in the "Outstanding Young Scientist of the 
Year" award from the Maryland Academy of 
Sciences. Dr. Leon Heppel's long and distin- 
guished work on nucleic acids resulted in the 
meritorious Service Medal of the Public Health 
Service. Superior Service Awards of the De- 
partment of Health, Education, and Welfare 
were received by Drs. N. R. Shulman, C. B. 
Anfinsen and J. E. Rail. 



The staff of an Institute, as has been noted 
in these reports before, are the very substance 
of the Institute. It is of interest, therefore, to 
note a gradual change that has come about at 
NIAMD as Clinical, Research and Staff Asso- 
ciate positions have become available for physi- 
cians, and the Staff Fellowship program has 
developed for scientists with a Ph.D. degree. 
Of a doctoral complement of some 234, the sen- 
ior permanent staff represent 140, or 60%. The 
Associates number 70 and Visiting Fellows and 
Scientists, on one or two year appointments, 
constitute 24. In addition to the above, the 
scientists in NIAMD have attracted 34 guest 
workers who came here with support from a 
wide variety of other organizations. The trend 
has, therefore, been towards the development 
of a larger and larger number of scientists 
here on a temporary basis. As science has be- 
come more complex, however, the time for the 
most profitable post-doctoral training has in- 
creased from one to 2 years and increasingly 
we see 3 years as a better period. The associate 
positions are generally for 2 years and the staff 
fellowship for 3 years. 

Concomitant with this change towards a 
larger temporary post-doctoral population has 
been the development of a more and more com- 
prehensive educational program. The graduate 
program at NIH lists 99 courses offered, rang- 
ing from cultural anthropology to chemical 
quantum mechanics. There are, in addition, 
several dozen tutorial seminars for research 
associates held each semester. Hence, the in- 
creased proportion of younger temporary in- 
vestigators at NIH has been accompanied by 
an increase in educational opportunities. These 
trends seem favorable omens for lively re- 
search. 



161 



162 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



OFFICE OF MATHEMATICAL RESEARCH 

Work has continued within the areas and 
along the lines indicated in previous summa- 
ries. While new studies, approaches and meth- 
ods have been initiated, in general the work of 
the past year can be subsumed under one or 
both of these categories: (i) generalization and 
extension in depth of ongoing studies in select- 
ed areas of mathematical biology and related 
mathematics; (ii) application, often concomi- 
tant with further development, of methods, 
models and theories previously developed to 
particular biological problems. 

A new theoretical approach has been devel- 
oped to define information content of a given 
set of data relative to a class of models. The 
method leads to criteria for the design of new 
experiments to select a given model or to im- 
prove estimation for a selected model. A meth- 
od of inference for assigning an individual to 
one of two alternative populations has been ex- 
tended to give an assignment to a hypothetical 
population "lying between" the two popula- 
tions thus yielding a quantitative measure 
which may be viewed as the "degree of transi- 
tion" of an individual from one population to 
the other. Revision and up-grading of the com- 
puter program (formerly NIH-0MR9B, now 
denoted SAAM) has continued. A program has 
been developed for the analysis of logical struc- 
ture of programs. Collaborative studies have 
continued in the following areas: Iodine kinet- 
ics, magnesium kinetics, and calcium kinetics 
(all in human subjects) and kinetics of iodina- 
tion of tyrosine in vitro. (Dr. M. Berman and 
Mrs. M. Weiss.) 

The development of a family of mathemati- 
cal models for the transport, diffusion and con- 
sumption of metabolites in the blood-capillary- 
tissue complex has been undertaken. The 
formulation is designed to take account of 
geometry of the arteriole-capillary-venule sys- 
tem, the flow and diffusion aspects, the kinetics 
of oxygen-hemoglobin association-dissociation 
and the kinetics of consumption in the tissue. 
Along with the analytical formulation compu- 
tational schemes are being developed capable of 
processing data arising from actual experi- 
ments of various types. For the particular 
geometry deemed appropiiate for skeletal 






muscle the analytical description yields a syS' . 
tern consisting of an elliptic partial differential f 
equation, a non-linear ordinary differential 
equation, a set of mixed boundary conditions 
and an interface condition. This system has 
been solved by constructing an iterative scheme 
and solving the partial differential equation 
through explicit formulation of this fundamen- 
tal solution. The programming of the iterative 
method for computer application must be pre- 
ceded by numerical-analytical studies which 
are under way. (Dr. J. Gonzales-Fernandez.) 

If a cell or tissue maintains a stationary in- 
ternal concentration of a metabolite which is 
different from that prevailing in the ambient 
medium and either no flow occurs or flow in 
fact occurs against an apparent gradient in 
concentration, then the possibility of active 
transport is entertained. In assessing such sit- 
uations, two experimental facts are often, or 
even always, relevant: (i) In general only the 
volume average of the internal concentration 
can be determined although the concentration 
is certainly non-uniform and direction of flow 
is determined at any point by the local gra- 
diant; (ii) Chemical or physical methodology is 
such that certain linear combinations of con- 
centrations are determined. The role of these 
two factors has been treated analytically on the 
basis of a rather general formulation of the ap- 
propriate diffusion-reaction problem. It has 
been shown that for a metabolite, consumed by 
one set of reactions and produced by another, 
appropriate separation of the sites of produc- 
tion and consumption can lead to apparent ac- 
tive transport, in that the average internal 
concentration may exceed that in the medium 
while flow occurs from medium into cell. A lin- 
ear combination of concentrations can likewise 
lead to active transport. This conclusion is but 
one of several corollaries of a series of theo- 
rems establishing properties of linear combina- 
tions of solutions of the diffusion-reaction 
equation appropriate to open metabolizing 
systems and which defy brief narrative sum- 
mary. (Dr. John Z. Hearon.) 

Mathematical and computer studies on visual 
systems have continued. Computation of the 
retinal local-averaging function was reported 
last year. The computed function has been ap- 
plied to square-wave data to show that the as- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



162 



similation effect in the perception of contrast 
observed by Helson is consistent with a local 
retinal averaging in conjunction with a "blur- 
ring function" which simulates optical imper- 
fections of the eye. Qualitative agreement be- 
tween predictions from the model and observed 
data is excellent and quantitative agreement 
can probably be improved by taking into ac- 
count the possible adaptive charges in the 
retinal averaging function. The Fuortes-Hodg- 
kin model for visual response of the Limulus 
eye has been extended and refined. Marked im- 
provement has been obtained by addition of a 
second feed-back loop and a different assign- 
ment of the source of the originally postulated 
feed-back loop. (Mrs. R. Marimont, Associate 
Member, OMR, NIMH.) 

Mathematical studies of dendritic neurons 
have been further developed with special refer- 
ence to a theoretical model for potentials in the 
olfactory bulb. Considerable effort has gone to- 
wards development and testing this model 
(this work was done in collaboration with Dr. 
G. Shepherd, at that time Associate Member, 
OMR, NINDB) . The study is designed to take 
into account a considerable amount of detailed 
anatomical and physiological information and 
to provide a theoretical method for interpreta- 
tion of recorded voltage patterns. Among the 
parameters or characteristics included are 
magnitude of geometric obstacle to invasion of 
soma and dendrites by antidi-omic impulse, 
magnitude of dendritic facilitation, kinetics of 
action potential and "activity" or "passivity" 
of the dendritic membrane. Computations from 
the mathematical description show the com- 
plicated interplay of these characteristics in 
determining whether an antidromic impulse 
upon reaching the soma will be blocked, be 
delayed or invade rapidly. Reasonably good fits 
to experimental data have been obtained and 
a paper is in preparation. (Dr. W. Rail.) 

It is now generally accepted that the ability 
of the mammalian kidney to produce concen- 
trated urine depends upon some type of 
counter current multiplier. One question has 
been whether active transport only in the thick 
portion of the ascending Henle's loop is sufii- 
cient for increasing sodiimi concentration in 
the inner medulla, if the parallel counter flow 
of the medullary vessels is taken into account. 



For a two-loop system, this question has been 
answered in the negative: It has been shown 
that the maximum interstitial sodium concen- 
tration in the outer medulla exceeds any con- 
centration in the inner medulla, both intersti- 
tial and counterflow loop. This conclusion has 
been illustrated by numerical solution for typi- 
cal cases and the work has been submitted for 
publication. This analysis has been extended to 
systems of any number of loops. By a rigorous 
but qualitative argument, it can be shown that 
a large class of systems cannot concentrate out- 
side the region of active transport. While the 
mechanism of renal concentration remains 
unspecified, these results, by ruling out a large 
class of systems, sharply narrow the range of 
possibilities. (Dr. J. Stephenson, Associate 
Member, OMR, NHI.) 

LABORATORY OF BIOPHYSICAL 
CHEMISTRY 

The main interest of this laboratory is the 
study of the relationship of the structure of 
biologically important macromolecules to func- 
tion. I would like to illustrate last year's activi- 
ties of this laboratory by examples. 

A special attention was paid to the interac- 
tion of the enzyme thrombin with fibrinogen. 
Thrombin is a highly specific proteolytic en- 
zyme which splits four peptide bonds between 
arginine and glycine residues in the fibrinogen 
molecule. In order to come to a better under- 
standing of this reaction, we modified the sub- 
strate as well as the enzyme molecules. 

Previously we have iodinated tyrosine resi- 
dues in the fibrinogen molecule and changed ly- 
sine residues into homoarginine. This year, 
fibrinogen was succinylated to change the 
charge characteristics of the molecule. In this 
case, it was found that thrombin released only 
peptide A from the modified fibrinogen, in con- 
trast to the other modifications. (Dr. J. 
Gladner and Dr. A. Osbahr.) 

Chemical modifications usually are very 
crude. One only occasionally succeeds in mod- 
ifying selected residues. We, therefore, de- 
cided to let nature do the modifications. From 
kinetic studies, we could convince ourselves 
that during evolution, both fibrinogen and 
thrombin underwent modification. In several 



164 



ANNUAL KEVIEW OF INTRAMURAL RESEARCH 



cases, the amino acid sequency of the peptides 
split off by thrombin from fibrinogen are also 
known. A few years ago, by using synthetic 
substrates for thrombin, we have found that 
portions of the substrate far removed from the 
bond split effected the reaction. From our kinet- 
ic studies, coupled with the known alterations 
nature introduced in the N-terminal portion of 
the fibrinogen molecule, we were able to deduce 
that residues removed from the bond split as 
far away as 18 residues have an effect on the 
enzyme. This shows that a comparatively large 
area of the enzjrme and the substrate come into 
contact during the enzymatic reaction. (Drs. K. 
Laki and N. Chandrasekhar.) 

When a protein is synthesized, its amino acid 
sequence is copied (Translated) from the nu- 
cleotide sequence of the messenger RNA. One 
of the problems is how then the newly synthe- 
sized peptide chain takes up its specific three- 
dimensional structure. One principle seems to 
be emerging: In watery milieu, hydrophobic 
residues crowd together and the hydrophylic 
residues come into the surface. It is quite rea- 
sonable to expect that these hydrophylic or 
polar groups may also form groups and cluster 
together. (Drs. H. Saroff and W. Carroll.) 

Dr. Saroff, in the case of ribonuclease, found 
evidence for the clustering of polar groups 
from ion-binding experiments. Because of the 
clustering, these polar groups collectively, and 
thus, more strongly, bind ions. 

Clustering of the polar groups of course puts 
great limitation on how the peptide chain can 
fold. From the consideration of these limita- 
tions, it was possible to propose a three-dimen- 
tional structure for the ribonuclease molecule. 
(Dr. H. Saroff.) 

The peptides released from fibrinogen have 
physiological activity. For example, Peptide A 
released from human fibrinogen acts as vaso- 
constrictor. It was important to know what 
portion of the peptide is responsible for this 
activity. By degradation studies, it was shown 
that the sequence Asp.-Ser.-Gly. in the peptide 
is responsible for the activity. (Drs. A. Osbahr, 
J. Gladner, K. Laki.) 

In muscular contraction, the structures we 
deal with are on an even higher level. Protein 
molecules in an ordered manner build up the 
contractile structure which, when the energy is 



supplied to it, can shorten and carry out work. 
In order to come to a better understanding of 
this process, chemical modifications were car- 
ried out on the proteins of the contractile 
structure. It was found that when the muscle 
fibers were acetylated or methylated, simple 
substances such as various salts brought about 
contraction. In these cases, it is reasonable to 
conclude that the binding of these ions is re- 
sponsible for supplying the energy necessary 
for the work carried out. 

There are indications that the binding of 
these substances leads to changes in the folding 
of the individual protein molecules that consti- 
tute the contractile structures. 

These studies are important because bio- 
chemistry is still unable to describe in detail, 
processes where one form of energy is con- 
verted to another one. (Drs. W. Bowen and D. 
Kominz.) 

LABORATORY OF EXPERIMENTAL 
PATHOLOGY 

Pathology of Rheumatic Disease 

An apparatus (arthrotripsometer) has been 
developed to determine in vitro the frictional 
characteristics of animal joints whose center of 
rotation varies throughout an oscillatory cycle. 
Unlike previously described studies, it provides 
values at all portions of the cycle of joint mo- 
tion instead of means. The instrument also 
senses the deformation of cartilage under dif- 
ferent conditions of joint motion and loading. 
The contours of the friction curves obtained 
from animal joints differed from those of a 
metallic (bronze on steel) journal bearing in 
having more conspicuous variation in the fric- 
tion coefficients at either end of the cycle of 
motion than did the latter. The relationship of 
the friction coefficients of the joints to varia- 
tions in load and frequency of motion differed 
from that of metallic bearings and is inconsist- 
ent with a simple boundary or hydrodynamic 
theory of joint lubrication. The data obtained 
in dog ankles and a metallic bearing indicate 
that: (1) animal joints had much smaller fric- 
tional losses than metallic bearings, even when 
the latter operated with excellent oil lubri- 
cants; (2) despite data presented by others on 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



165 



artificial bearings, synovial fluid proved to be 
an effective lubricant in joints. Under condi- 
tions of motion restoration of compressed joint 
cartilage to its normal thickness proceeded 
much more rapidly than when there was no mo- 
tion. (Mr. Frank C. Linn.) 

Dietary supplements of saturated fats, either 
in the form of lard or hydrogenated sai!lower 
oil, had no demonstrable deleterious effect on 
the development of degenerative joint disease 
in male DBA/2JN mice. Although the lard re- 
sulted in greater obesity than did the cotton- 
seed oil supplement, the weight gain was more 
rapid and greater when safflower oil was used 
rather than 2 hydrogenated products of saf- 
flower oil having iodine numbers equal to that 
of the cottonseed oil and lard, respectively. 
(Drs. Sokoloff and Mickelsen.) 

Scientific Literature Retrieval 

The urgent need for development of ade- 
quate methods for retrieval of scientific litera- 
ture as a research tool prompted a literature 
analysis project to be carried out for the Amer- 
ican Rheumatism Association in this labora- 
tory. Three steps were taken to improve the 
dissemination and retrieval of information on 
rheumatic diseases: (1) the domain of rheuma- 
tology and the limits to which basic research 
literature should be included in its biblio- 
graphic services were delineated by a ques- 
tionnaire survey; (2) a novel Thesaurus of 
Rheumatology was prepared to improve the 
indexing of publications and to codify the 
vocabulary; (3) a computer based semimonthly 
publication, the Index of Rheumatology, was 
developed with the cooperation of the National 
Library of Medicine. This bibliography is ex- 
pected to yield approximately 6,000 indexed 
citations per year. (Miss Ruhl and Dr. 
Sokoloff.) 

It was shown statistically that identification 
codes for technical (or any other) documents 
could be assigned at random by the publisher 
without causing too many cases of confusion. 
The principal virtue of this method, aside from 
its simplicity, would be its universality ; no con- 
vention or centralized authority for assigning 
code numbers would be required. (Dr. McCut- 
chen.) 



Chromosome Studies in Chronic Myelogenous 
Leukemia 

Seventy-three patients referred to or attend- 
ing the clinics of the Clinical Center of the 
N.I.H. with the diagnosis of chronic myelogen- 
ous leukemia (CML) between May 1961 and 
October 1963, have been studied as to the sig- 
nificance of the so-called Ph^ (Philadelphia) 
chromosome in the marrow cells with respect 
to the state of the disease. Sixty of the 73 pa- 
tients were Ph^ chromosome positive and 13 
were negative. The 13 Ph^ negative cases dif- 
fered from the Ph^ positive group by having 
lower white blood cell and platelet counts and 
by having a larger proportion of young chil- 
dren under the age of seven. However, the 
most significant difference was the finding that 
the Ph^ negative patients responded poorly to 
chemotherapy and had a median survival of 18 
months as compared to 45 months for the Ph^ 
positive population. (P<0.1). The Ph^ chro- 
mosome was observed during all phases of the 
disease and was unaffected by treatment. In 
20% of the Ph^ positive patients, aneuploidy, 
mainly hyperdiploid Ph^ positive cells, was 
also observed. Two patients had cell lines with 
2 Ph^ chromosomes. In the Ph^ negative 
group, there was only one patient with marked 
aneuploidy. The rest had apparently normal 
karyotypes in their marrow cells. (Dr. Tjio.) 

Characterization and Quantitation of Enzymes 
in Histochemieal Systems 

The parameters necessary for quantitation 
in a histochemieal system and for relating the 
histochemieal and biochemical systems have 
been the subject of continued study. In an 
effort to determine the relationship of the sol- 
uble and tissue-bound (so-called "lyo" and 
"desmo") components of enzymes in histochem- 
ieal and biochemical systems, an investiga- 
tion of the hydrolysis of L-leucyl ^-naphthyla- 
mide in rat kidney was undertaken. It was 
found that soluble enzymes were completely ex- 
tracted from the tissue section during a ten 
minute incubation. By ultracentrifugation, 
fractionation procedures and DEAE column 
chromatography the soluble enzyme was en- 
riched and found to be inhibited by 2J-chloro- 
mercuribenzoate and reactivated by cysteine. 



166 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



The tissue-bound enzyme was solubilized and 
purified over 1,000 fold by autolysis, fractiona- 
tion procedures and DEAE column chromatog- 
raphy and its molecular weight and amino- 
acid composition determined. Electrophoresis 
of this preparation revealed only two protein 
bands, both enzymically active. These were 
separated by elution and found to be isozymes. 
The tissue-bound enzyme was activated by 
Co+^ and inhibited by o-phenanthroline, and 
hydrolyzed peptides, polypeptides and amino 
acid /3-napthylamides at rates dilfering mark- 
edly from those of the soluble enzyme and the 
classical leucine aminopeptidase of Smith. A 
study of these hydrolytic rates revealed it to 
have the functional capacity of an "aminopoly- 
peptidase." By the use of a recording micro- 
densitometer and constant-flow incubating 
chamber mounted on the microscopic stage, the 
enzyme in the histochemical section was shown 
to have the same salient characteristics as 
those of the purified tissue-bound enzyme. It 
was concluded that in the system studied the 
"lyo" and "desmo" components represented in 
reality distinct enzymes with exclusive charac- 
teristics. The fresh frozen section used his- 
tochemically appeared to be comparable to the 
particulate fraction of a 105,000 Xg homogen- 
ate ultracentrifugate. Zymograms of the whole 
homogenate demonstrated prominently the sol- 
uble enzyme. These studies are preliminary to 
the comparative evaluation of enzyme reaction 
characteristics in a histochemical system with 
enzymes previously characterized by bio- 
chemical techniques. (Drs. Felgenhauer and 
Glenner.) 

In addition, by the synthesis and utilization 
of a wide variety of amino acid and dipeptide 
;8-naphthylamides (Drs. Glenner, Folk, and Co- 
hen) , it has been possible to follow by a simple 
colorimetric assay procedure the purification of 
a previously undescribed aminopeptidase. This 
enzyme, termed, aminopeptidase B, has been 
purified 1,000 fold and has been found to hy- 
drolyze selectively N-terminal L-lysine and 
arginine residues from peptides and polypep- 
tides. The enzyme is found in the soluble frac- 
tion of rat tissue homogenates of duodenum, 
liver, and pancreas and has been demonstrated 
to be inhibited by j9-chloromercuribenzoate and 



reactivated by sulfhydryl compounds, e.g., 
cysteine and glutathione. 

The biochemical concept of lysosomes (cyto- 
plasmic bodies containing hydrolytic enzymes) 
has been the subject of extensive electron mi- 
croscopic-histochemical investigation using as 
a marker for cytosome (lysosome) demonstra- 
tion, the sites of hydrolysis of ^S-glycerophos- 
phate at acid pH. A method has been derived 
for the successful electron miscoscopic localiza- 
tion of a further "lysosomal" hydrolytic enzy- 
mic activity, that of aryl-sulfatase. Using bar- 
ium ion as the capture reagent to precipitate 
sulfate ions released during enzymic hydrolysis 
of the aryl ester, we have been able to demon- 
strate the localization of this activity (the 
type of aryl-sulfatase responsible is as yet 
unknown) to single membrane bodies corres- 
ponding to cytosomes (lysosomes) and cytopha- 
gosomes. This represents the first evidence con- 
firming the existence of a "lysosome" enzyme 
other than acid phosphatase to the cytoplasmic 
bodies described collectively as lysosomes. In- 
vestigations are presently in progress to deter- 
mine whether the same lysosomes contain both 
hydrolytic enzymes. (Drs. Hopsu and Glenner.) 

Human Lymphocytes in Tissue Culture 

MacKinney, Stohlman, and Brecher had 
shown that when normal human blood is grown 
in tissue culture with phytohemagglutinin, 
small lymphocytes can transform into large 
"blast-like" cells capable of DNA synthesis and 
division. The present studies were designed to 
examine the events which precede mitosis of 
these cells in culture. Combined isotopic and 
radioautographic techniques revealed that 
RNA synthesis occurred very soon after the 
initiation of cultures and preceded DNA syn- 
thesis by 24 hours. A marked increase in protein 
synthesized by the transforming cells was ob- 
served in the first 48 hours of culture. The ma- 
jor portion of this newly synthesized protein 
migrated electrophoretically as alpha globulin; 
only a minor fraction of it migrated as beta or 
gamma globulin. Studies are in progress to 
identify immunoelectrophoretically the specific 
proteins synthesized and to determine whether 
their synthesis is a necessary requisite for divi- 
sion. (Dr. Epstein.) 



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NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



167 



RNA Metabolism in Relation to Steroid Admin- 
istration 

Steroid hormones are known to cause an in- 
crease in ribonucleic acid and in protein syn- 
thesis. Histochemical investigation of rabbit 
uterus reveals an increase in the basophilic 
chromidial substance attributable to an in- 
crease in RNA in stromal and smooth muscle 
cells of the rabbit uterus following treatment 
with steroids. (Drs. S. N. Cohen [LGAR- 
NIAID], S. S. Spicer and K. L. Yielding.) 

Nucleic Acid and Protein Synthesis 

Cytochemical and quantitative autoradio- 
graphic studies on the morphology and function 
of the nucleolus in nucleic acid and protein syn- 
thesis have been conducted with HeLa cell cul- 
tures. It was found that H^-lysine is rapidly 
incorporated into nucleolar material solubilized 
either by agents which hydrolyze RNA or by 
the proteolytic enzyme trypsin. Concentrations 
of Actinomycin D which inhibit the synthesis 
of nucleolar RNA also inhibit the incorporation 
of lysine into nucleolar material but not into 
extranucleolar chromatin and cytoplasm. When 
stained for e-amino groups of basic protein 
with Biebrich scarlet under selective conditions 
of fixation, nucleoli showing lysine incorpora- 
tion present a characteristic granular substruc- 
ture not exhibited or severely altered in cells 
treated with actinomycin or under conditions 
of RNA extraction. These findings are inter- 
preted as evidence for the synthesis in the nu- 
cleolus of a lysine containing protein which is 
linked metabolically or chemically to RNA and 
appears to be independent of chromosomal and 
cytoplasmic sites of RNA synthesis. 

Preliminary results of quantitative autora- 
diographic experiments with H'-lysine, H^-ar- 
ginine, or H^-uridine-labeled HeLa cells pre- 
treated with puromycin and Actinomycin 
indicate that this nucleolar protein is ribosomal 
in type rather than transfer RNA and that it is 
demonstrable autoradiographically only under 
defined conditions of aldehyde fixation. Fixa- 
tion at suboptimal conditions of temperature, 
pH and ionic strength or hydrolytic removal of 
RNA results either in solubilization of some 
peptide groups in aqueous media or in redistri- 



bution in non-nucleolar areas of the nucleus. It 
is hoped that this type of investigation may 
contribute in relating morphologic techniques 
in pathology and cytology to the problems of 
molecular biology. (Dr. Suskind.) 

An extension of these investigations, an au- 
toradiographic, microspectrophotometric and 
virologic study, designed to ascertain the sites 
of viral nucleic acid synthesis and viral protein 
synthesis of Rous sarcoma virus in trans- 
formed (neoplastic) and non-transformed tis- 
sue culture cells, is in progress. (Drs. Suskind 
and Rabotti [LVO-NCI].) 

Soluble Ribonucleic Acid (s-RNA) 

In collaboration with Dr. Bruce N. Ames 
(LMB-NIAMD) a technique has been devel- 
oped for separation of the various amino acid 
acceptor species of s-RNA by chromatography 
on DEAE-sephadex. The technique provides as 
good separation as has been obtained by DEAE 
chromatography in urea or by gradient parti- 
tion chromatography, and it is more versatile 
and convenient than these methods. It is ex- 
pected that the new chromatographic method 
will soon be available for the preparation of 
single s-RNA species and for the analysis of 
changes occurring in s-RNA which can be re- 
lated to alterations in the rate and pattern of 
protein synthesis. (Dr. Smith.) 

DNA in Crithidia 

In view of the current interest in cytoplas- 
mic organelle replication, a collaborative study 
was initiated on a hemoflagellate, Crithidia sp. 
Crithidia were incubated with H^-thymidine 
for 24 hours and then fixed and prepared for 
electron microscope autoradiography. A Chalk- 
ley grid analysis of the electronmicrographs in- 
dicated that the H'-thymidine uptake by the 
kinetoplast, a specialized mitochondrial organ- 
elle, was about three times as great per unit 
random cross-sectional area as that by the nu- 
cleus. Practically no background label is found 
with this technique. These findings add strong 
evidence that substantial DNA synthesis may 
occur outside of the nucleus. (Drs. Wetzel, 
Luke and Greenblatt [LPB-NIAMD]. 



168 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Phosphatases on E. coli 

Cytochemical and morphological studies on 
E. coli have revealed the presence of various 
phosphatases in different locations within the 
bacteria. Thus, non-specific alkaline phospha- 
tase has been localized on the surface of the cell 
in some strains whereas enzymatic hydrolysis 
of cyclic diester phosphates has been visualized 
in areas beneath the cell wall. (Drs. B. K. Wet- 
zel, S. S. Spicer and L. A. Heppel [NIAMID- 
LBM]. 

Histidinol Phosphate Phosphatase 

The localization of the three phosphate split- 
ting enzymes of the histidine biosynthetic 
pathway in Salmonella typhimurium by elec- 
tron microscopy could provide information 
about the compartmentalization of the path- 
way within the cell. Moreover, the histidine 
pathway in bacteria appears to be a good sys- 
tem for studying possible improvements in ex- 
isting phosphatase localization techniques. The 
activity of one of the enzymes, histidinol phos- 
phate phosphatase, has been studied in vitro 
under conditions required for phosphate trap- 
ping on the microscopic level and for fixation 
of the cells, and an optimal procedure has been 
established by which about 15% of the phos- 
phatase activity is preserved. Electron micro- 
scopic studies are currently in progress. (Drs. 
Smith, Wetzel and Spicer.) 

The interaction of mitochondria and an iso- 
lated mitochondrial protein with phenols that 
uncouple oxidative phosphorylation has been 
studied. It was concluded that lipophilic rea- 
gents such as thyroxine are bound to the pro- 
tein moiety of mitochondria and are not simply 
dissolved in the lipid fraction as had been 
widely assumed. Oxidative phosphorylation can 
be restored by the addition of serum albumin, a 
protein which more tightly binds the reagents 
and thereby removes them from mitochondria. 
(Drs. Brabus and Weinbach [LPD-NIAID]. 

Localization of Antigens with Antibody Tech- 
niques 

The ferritin labeled antibody technique for 
visualizing specific antigens in cells at the elec- 
tron microscope level of resolution has been ap- 



plied to investigation of the fine structural 
location of the antigen responsible for the cir- 
culating antibody present in patients with 
myasthenia gravis. This technique has shown 
localization of ferritin labeled antibody to the 
H-zone of rat striated muscle. However, the 
binding of a variety of control conjugates casts 
doubt on the immunological specificity of this 
observation and raises question as to the pres- 
ence in the H-zone of a protein with unique 
reactivity for ferritin conjugated protein. The 
relationship of this staining to the immuno- 
specific staining of striated muscle, observed 
with fluorescein conjugated antibodies from 
sera of myasthenia gravis patients remains to 
be investigated. (Drs. S. D. Douglas, A. J. 
Gottlieb [LMB-NIAMD], A. J. L Strauss 
[DB-NCI], and S. S. Spicer.) 

Fluorescein 

By means of the Coons' fluorescein labeled 
antibody technique it has been possible to 
demonstrate the presence of prolactin in rat pi- 
tuitary tumors. Fluorescein labeled anti-prolac- 
tin also stains prolactin of glands in fish pitui- 
tary. The significance of this observation is 
under investigation. In an extension of the proj- 
ect on visualization of cell antigens by fluores- 
cein labeled antibodies, the site of biosynthesis 
of growth hormone is being investigated in the 
pituitary of normal mice and dwarf mice. Fluo- 
rescein labeled antibody to glycerophosphate 
dehydrogenase has also been employed in stain- 
ing heart muscle and shows the distribution of 
this enzyme in cardiac muscle fibers. (Dr. E. 
W. Emmart.) 

Fluorescein labeled antibodies to blood group 
antigens are currently being employed as a 
means of demonstrating blood group sub- 
stances in mucus secreting epithelia and con- 
nective tissues. Comparison of these results 
with those of other histochemical staining 
procedures is being determined to evaluate the 
histochemical nature of the blood group reac- 
tive substances. (Drs. Douglas and Spicer.) 

Carbohydrate Histochemistry 

A histochemical terminology for mucosub- 
stances has been developed along with a clas- 
sification of types of mucosaccharides which 



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169 



j can be distinguished histochemically. (Drs. 
I Spicer, Leppi and Steward. ) Current efforts in- 
! elude development of methods for differentiat- 
[ ing mucosubstances in tissue sections by light 
! microscopy. A recently developed formazan 
method reveals differences in mucosubstances 
not previously observed with conventional PAS 
and other histochemical procedures. The basis 
for this differentiation is under investigation. 
Development of additional methods for selec- 
tively localizing carbohydrates and other com- 
ponents in tissue sections are also in progress. 
(Dr. Stoward.) 

Methods have been developed for specifically 
identifying sulfated mucosubstances histo- 
chemically, for differentiating periodate re- 
active from periodate unreactive mucosub- 
stances, and for demonstrating differences be- 
tween mucosaccharides on the basis of suscepti- 
bility of enzymatic digestion. These methods 
have been applied to an investigation of the 
types of mucosubstances present in salivary 
glands of primates and ungulates. The marked 
heterogeneity of the various types of secretions 
in these glands has been recorded and partial 
characterization of the diverse components has 
been accomplished including particularly dem- 
onstration of periodate reactive sulfomucin in 
certain epithelia. (Drs. Spicer and Leppi.) 

Attempts to specifically visualize acid muco- 
substances at the fine structural level have led 
to the development of a method for staining 
sialomucins and sulfomucins in mouse epithe- 
lia, in addition to the visualization of some con- 
nective tissue mucosubstances. This technique 
involves treatment of sections for electron mi- 
croscopy with various solutions of ferric chlo- 
ride at controlled pH levels and provides selec- 
tive staining of acid mucosubstances in tissue 
prepared for electron microscopy. (Mrs. Wet- 
zel, Drs. Wetzel and Spicer.) 

A battery of histochemical methods has been 
applied to investigation of the types of secre- 
tion in patients with mucoviscidosis (cystic 
fibrosis) and the presence of sulfomucins as 
well as sialomucins in the abundant secretions 
of these patients has been demonstrated. It has 
also been found that secretions in cystic 
fibrosis appear to differ only quantitatively and 
not qualitatively from those in normal controls 



resembling closely those in other hypersecre- 
tory states. (Drs. Lev and Spicer.) 

Application of methods for carbohydrate 
histochemistry to an investigation of connec- 
tive tissue mucosubstances has shown a variety 
of biochemically unrecognized mucosubstances 
in various connective tissue sites and in par- 
ticular has differentiated several mucosub- 
stances in the outer, visual layer of the retina. 
This investigation also has shown precise lo- 
calization of hyaluronidase resistant mucopoly- 
saccharide (keratan sulfate?) in cartilage, 
together with an interesting, intimate morpho- 
logic relationship of this mucopolysaccharide 
with sites or calcification. (Drs. Spicer, Horn 
and Leppi.) 

Investigation of the validity of hyaluroni- 
dase digestion as a means of localizing muco- 
polysaccharides histochemically has shown that 
because of contaminating protease the results 
are of questionable value when various com- 
mercial enzymes are used. (Drs. Leppi and 
Stoward.) 

Histochemistry of Mucopolysaccharides 

The epithelium lining the distal bronchial 
tree and the mesothelium of the pleural surface 
were studied histochemically for mucopolysac- 
charides in rabbit, Syrian hamster, mouse, 
guinea pig and man. In the larger more proxi- 
mal bronchial radicles, large quantities of sul- 
fated mucopolysaccharides predominated. In 
contrast, sialomucins in increasing amounts 
were found more distally along the bronchiolar 
and alveolar surfaces and the pleural mesothe- 
lium. The sialomucin in the distal bronchial 
tree was increased in the presence of acute in- 
flammation and was absent when chronic in- 
flammation or pulmonary edema was present. 
It is postulated that the sialomucinous material 
may have biologic significance because of its 
bacteriostatic and possible pulmonary surfact- 
ant properties. (Drs. Luke and Spicer.) 

In an earlier study, evidence was found that 
a sulfated mucosubstance is a constituent of 
the fibrinoid thrombi of the generalized 
Shwartzman reaction, and it was postulated 
that this mucosubstance contributes to the 
thrombotic process and may be derived from a 
sulfated mucopolysaccharide originating in 



170 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



azurophilic granules of heterophil leukocytes. 
(Drs. Horn and Spicer.) More recently, a study 
was initiated on the replication pattern of the 
several cytoplasmic granules in the granulocyte 
serizs. Rabbit bone marrow was incubated in 
vitro with S^^-sulfate for varying periods of 
time and then fixed, sectioned and prepared for 
electron microscope autoradiography. A pre- 
liminary study indicates that the localization of 
the isotope in heterophil myelocj'-tes occurred 
initially in the Golgi region and subsequently 
in azurophilic type granules. (Drs. Horn, Luke, 
Spicer, and Wetzel.) 

Electron Microscopy 

Thyroid gland. A protracted morphological 
and cytochemical study of rat thyroid glands 
following administration of thyroid stimu- 
lating hormone (TSH) has been concluded. 
Colloid resorption appears to proceed through 
the phagocytic engulfment of colloid droplets 
by apical pseudopods and their subsequent ac- 
quisition of acid phosphatase activity. Small, 
dense, enzyme-rich granules appear to provide 
the acid phosphatase through fusion with 
newly formed colloid droplets within 15 to 30 
minutes after TSH stimulation. Synthesis of 
thyroglobulin would appear to proceed via a 
separate mechanism involving small (0.1^) ves- 
icles prominent in the Golgi region and be- 
neath the apical plasma membrane of some but 
not all follicular cells in a given specimen. 
These studies represent an extension of investi- 
gations at the light microscope level which 
showed engulfment of luminal colloid into thy- 
roid epithelial cell droplets and admixture of 
both acid phosphatase and esterase with these 
droplets shortly after stimulation. (Drs. Wet- 
zel, Spicer and S. H. Wollman [LPHY-NCI].) 

Myeloid elements. Studies on the fine 
structure and cytochemistry of rabbit myeloid 
elements are near completion with the goals of 
more thoroughly characterizing granulocyte 
development and contributing to an under- 
standing of the nature of their cytoplasmic 
granules. Rabbit heterophils appear to contain 
at least three distinct types of cytoplasmic 
granules. The primary granule, rich in acid 
phosphatase, and (by correlative histochemical 
studies) sulfated mucosubstances and basic 



proteins, is produced by the earliest identifiable : 
heterophils. Application of electron microscope ! 
autoradiography also has afforded a means of | 
confirming the localization of the sulfated mu- 
copolysaccharide in the primary type of gran- 
ule in rabbit heterophils. Numerous primary 
granules accumulate as the cells enlarge, but an 
abrupt shift seems to occur to de novo syn- 
thesis of alkaline phosphatase-rich secondary 
granules. Continued cell division probably ac- 
counts for the diminished numbers of primary 
granules present in more mature cells, which 
contain numerous secondary granules and an 
additional population of smaller, acid phospha- 
tase-rich tertiary granules. A complimentary 
combination of morphological and cytochemical 
characteristics permits this type of analysis. 
Differing roles of acid phosphatase in granule- 
genesis are evident in eosinophiles, basophiles, 
mononuclear cells and platelets. Acid phospha- 
tase-rich organelles, presumably functioning as 
cytolysomes, have been encountered in ersrth- 
roblasts and reticulocytes at the time of resorp- 
tion of the chromidial substance and the mito- 
chondria of these cells. Cytochemical studies of 
heterophils phagocytosing bacteria have dem- 
onstrated the contribution of all three types 
of granules and their phosphatases to the bac- 
teria-laden phagocytic vacuoles. (Drs. Wetzel, 
Horn, Luke, and Spicer.) 

Epinephrine 

It was shown previously that exposure of an- 
imals to various stresses such as high altitude 
hypoxia, cold or arduous exercise produces 
liver glycogen depletion, fatty changes in the 
viscera and elevations in certain serum en- 
zymes. Since release of epinephrine and nore- 
pinephrine from the adrenal medulla is be- 
lieved to play a role in the response to such 
stresses, the effects of epinephrine in oil, which 
is slowly absorbed and has a prolonged action, 
were studied. 

Rats given a large dose of epinephrine in oil 
subcutaneously developed marked transient 
fatty changes in heart, liver, kidney and 
muscle, severe myocarditis, pulmonaiy hemor- 
rhages with hemoglobin-like crystalline de- 
posits, and renal hemoglobin casts. Liver glyco- 
gen was depleted. Serum values of glutamic 



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171 



oxalacetic and pyruvic transaminases, aldolase 
and urea nitrogen rose sharply and alkaline 
phosphatase fell 24 hours after a dose and 
returned to normal within 72 hours. Pretreat- 
ment with Dibenamine hydrochloride, an 
adrenergic blocking agent, prevented or les- 
sened most pathologic and serum enzyme 
changes, but not depletion of liver glycogen. 

It was concluded that the serum enzyme 
changes are due to alterations in cellular per- 
meability. This altered permeability is attrib- 
uted to a relative hypoxia, due to changes in 
oxidative metabolism with increased oxygen 
consumption by tissues induced by epinephrine, 
and to severe local hypoxia, caused by pro- 
longed marked vasoconstriction. The fatty 
changes, particularly in the heart, are attribut- 
ed to the effects of epinephrine on fat mobiliza- 
tion and metabolism and the specific avidity of 
the myocardium for storing catechol amines. 
(Drs. Highman, Altland [LPB-NIAMD], and 
Garbus.) 

Erythropoietin Production by Juxtaglomerular 
(JG) Cells 

A study of rats exposed to high altitude has 
revealed additional evidence that the renal JG 
cells produce erythropoietin. Various investiga- 
tors have sought such a relationship, but their 
methods of inducing oxygen deficiency also de- 
creased renal blood volume so that the observed 
results could have stemmed from either factor. 
In the present study, rats were exposed to a 
simulated altitude of 25,000 feet 6 hours daily 
and 5 days a week for 6 weeks. This resulted in 
polycythemia without a decrease in renal blood 
volume. Rats were killed either before or 
immediately after altitude exposure and the de- 
gree of granularity of the JG cells or juxta- 
glomerular cell index (JGI) was determined by 
the method of Hartroft and Hartroft. Plasma 
erythropoietin levels were determined by the 
bioassay method of Stohlman, and an improved 
method of extracting erythropoietin from the 
kidney was devised. 

A close correlation was found between the 
granularity of the JG cells and circulating 
erythropoietin levels. Thus, after a weekend of 
rest and before exposure, the JGI was high and 
the circulating erythropoietin levels were low. 



Following exposure, however, large decreases 
in the JGI were accompanied by high circulat- 
ing erythropoietin levels. Similar correlations 
between extractable renal erythropoietin and 
the JGI were observed. 

These findings support the view that JG cells 
are concerned with production of erythropoie- 
tin and that their increased secretory activities 
play a role in acclimatization to high altitude 
by increasing the circulating red cell mass 
through liberation of increased amounts of 
erythropoietin. (Drs. Demopoulos, Highman, 
Altland [LPB-NIAMD], Gerving [Univ. of 
Southern Calif.], and Kaley [N.Y. Univ.].) 

Effect of Adrenalectomy on Work Performance 

In collaboration with Drs. Maling (LCP- 
NHI), Altland and associates, studies are near- 
ing completion comparing biochemical and 
histopathologic changes during exercise be- 
tween normal intact rats and adrenalectomized, 
adrenal demedullated and chemically sympa- 
thectomized rats. Sympathectomy was produced 
in adrenal demedullated rats by depleting the 
stores of catechol amines with syrosingopine or 
by blocking release from nerve endings with a 
bretylium-like drug, BW 392C60. Running in a 
rotating cage, intact rats did not become fa- 
tigued in 4 hours, whereas the other groups be- 
came fatigued within 90 to 200 minutes. Differ- 
ences between groups were noted in changes 
during exercise in levels of plasma glucose, 
free fatty acid and lactic acid as well as in the 
triglyceride and glycogen content of various 
tissues, using both chemical and histologic 
methods. The differences suggest that the in- 
creased susceptibility to fatigue of adrenalecto- 
mized and sympathectomized animals is due to 
impairment in their ability to mobilize ade- 
quate energy fuel. (Dr. Highman.) 

Dipetalonema witei 

In a collaborative study with Dr. Weinstein 
(LPD-NIAID), it was found that following 
subcutaneous infection of the jird, a rodent 
found in North Africa, with Dipetalonema wi- 
tei (a filaria) larvae dissected from a tick, Or- 
nithodorus tartakovski, extensive nervous sys- 
tem symptoms developed in some of the ani- 
mals. The symptoms ranged from severe limb 



172 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



incoordination, spastic movements and partial 
paralysis to complete paraplegia, between 31 to 
93 days following infection. Worms (filaria) 
were found by gross dissection and histopatho- 
logic examination in the brain and spinal cord 
of some of these animals as well as in various 
other tissues. A histopathologic study is near- 
ing completion to determine how the filaria 
reach the central nervous system from the site 
of inoculation in the subcutaneous tissue of the 
abdominal wall. Although it is not known 
whether this parasite is pathogenic for man, 
this study may be important in elucidating the 
mode of entry of other parasites that attack 
the central nervous system in man. (Dr. High- 
man.) 

Toxic Compounds 

It has been proposed that the more stable io- 
date salts be substituted for the iodide salt for 
use in the prevention of goiter in tropical coun- 
tries. A study was made with Dr. Webster 
(LBP-NIAMD) on the acute and subacute tox- 
icity of potassium iodate in dogs. In dogs that 
died or were killed within 24 hours after a 
large oral dose of potassium iodate, 200-250 
mg/kg, there were noted areas of hemorrhagic 
necrosis in and near the antrum of the stom- 
ach, fatty changes in the liver and kidney, de- 
pletion of lipid in the adrenal cortex, and less 
constantly, centrilobular necrosis in the liver 
and foci of necrosis in the renal cortex and 
bladder mucosa. One dog, killed about 10 weeks 
after a dose of 200 mg/kg, showed degenera- 
tive changes in the retina. Subacute toxicity 
was studied in 4 dogs given repeated doses of 
6-100 mg/kg KIO3 for 68-192 days. There 
were large deposits of hemosiderin, particu- 
larly in the spleen, suggesting hemolytic ac- 
tivity by iodate, and a slight to moderate de- 
crease in red and white blood cell counts. Two 
dogs that received the last dose of iodate on the 
day of sacrifice showed foci of hemorrhagic ne- 
crosis in the gastric mucosa as described above. 
No retinal degeneration or other significant 
changes were noted. A report on the chronic 
oral toxicity of potassium iodate in dogs will 
follow. A report on these studies was submitted 
earlier to the Food and Drug Administration. 
(Dr. B. Highman.) 



Cycasin Research 

Experimental studies with the glycoside, cy- 
casin, a j8-D-glucosyl-oxyazoxy-methane, isolat- 
ed from the seeds of C. circinalis has continued 
during the past year. The glycoside is highly 
toxic in rats when taken orally in large 
amounts producing massive liver necrosis. It is 
carcinogenic when consumed in small amounts 
by conventional laboratory animals. Studies in 
rats indicate that the carcinogenic effect is not 
limited to one specific site, but that one is deal- 
ing with a general carcinogen producing neo- 
plastic disease at various sites simultaneously 
in a respectable percentage of the animals. 
Although the development of the acute toxic 
effects following ingestion is well established 
for man, it is not known at present whether it 
is carcinogenic for man. 

Recent studies by Dr. Matsumoto indicate 
that cycasin methylates nucleic acids, and 
methylation of the guanine in the 7 position 
has been indicated. Its mutagenic effect is 
briefly described by Dr. Smith below. Thus far 
no evidence has been obtained that it is terato- 
genic. Its radiomimetic effect has recently been 
noted by Dr. Teas. 

During the past year, data have been collect- 
ed along two main lines of investigation. One 
dealt with the carcinogenic effect of cycasin 
after short- and long-term exposures of rats to 
the glycoside ; the other with the excretion pat- 
tern of cycasin in germ free and conventional 
rats, a project summarized below by Dr. Spatz. 

A comparative study between short- and 
long-term exposures of rats to cycasin has 
yielded the information that long-term expo- 
sures (9 months) produced a high incidence of 
hepatomas followed by renal and intestinal 
neoplasm in frequency. On the other hand, 
short exposures (13-21 days) to cycasin pre- 
dominantly induced kidney tumors followed by 
hepatic and intestinal neoplasms in frequency. 
An analysis of these neoplasms is in progress, 
but two aspects of particular interest might be 
mentioned. Among the kidney tumors, neph- 
roblastomas, tumors comparable to Wilms' tu- 
mors in children, have been observed, and it 
was found furthermore that such tumors could 
be induced most readily when weanling rats 
were started on cycasin. The same type of tu- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



173 



mor only rarely occurred in rats placed on a 
cycasin diet after attaining sexual maturity. 
The possibility that chemical carcinogenesis at 
times may be instrumental in the development 
of Wilms' tumors suggests itself, and several 
studies are under way to test this by exposing 
rats at various times during pregnancy to cyca- 
sin and observing their offsprings (with Dr. 
Spatz). The histogenetic background of the 
nephroblastomas is presently investigated in 
collaboration with Dr. Gusek. 

The intestinal tumors have uniformly oc- 
curred in cecum and colon and, as a rule, have 
been characterized by abundant mucus produc- 
tion whether they were polypoid or broadly in- 
filtrating carcinomas. Studies involving bacte- 
rial flora of various segments of the alimentary 
tract, in particular in relation to their ability 
to cleave the glycoside, are being started in the 
hope to learn more about the site of liberation 
of the aglycone from the glycoside. This type of 
study, theoretically at least, offers an oppor- 
tunity to examine one possible cause for about 
10 percent of the rats not having developed 
neoplasms. Cycasin, unless cleaved in the in- 
testinal tract by a jS-glucosidase of bacterial 
origin is excreted unchanged without produc- 
ing toxic effects. The bacterial flora deter- 
mines, therefore, whether liberation of the tox- 
in and carcinogen occurs. (Dr. Laqueur.) 

Excretion of Ingested Cycasin in Germ-free and 
Conventional Rats 

The glycoside, cycasin, a /3-D-glucosyl-oxy- 
azoxymethane, had been shown to be hepatotox- 
ic and carcinogenic in rats, guinea pigs and 
mice. Investigations in vivo by Dr. Matsumoto 
demonstrated that the aglycone of cycasin pro- 
duced acute and chronic manifestations of tox- 
icity similar to those described with cycasin. 
The absence of acute toxic effects following 
either intraperitoneal injection of cycasin or 
after oral administration of cycasin in germ 
free rats, suggested that cycasin was degraded 
to the aglycone in the alimentary tract of ani- 
mals. An investigation into the fate of ingested 
cycasin in germ free and conventional rats was 
undertaken, and the time required for the dis- 
appearance of cycasin in urine and feces after 



discontinuation of cycasin feeding was ex- 
amined. 

Germ-free rats excreted 60 to 97 % of ingest- 
ed cycasin in urine and a trace to 32 % in feces, 
while the conventional animals excreted 18 to 
32% of ingested cycasin in urine and to 5% 
in feces. The percent of unaccounted cycasin in 
germ free rats was to 8% while in conven- 
tional animals, it was 65 to 82% of the ingest- 
ed cycasin. It is assumed at present that the 
amount of cycasin unaccounted for reflects that 
part of the ingested cycasin which has been 
metabolized by the animal. This is supported 
by other observations in which the severity of 
the liver injury roughly paralleled the amount 
of cycasin metabolized. 

The residual excretion of cycasin in the first 
24 hours after the animals were returned to 
the basal diet was to 1% in urine of both 
germ-free and conventional rats. In the feces, 
germ-free animals excreted 0.3 to 0.7%, while 
none was found in conventional animals. 
During the second 24 hours, cycasin was no 
longer found in the urine and feces of either 
group. 

These results indicated that cycasin was not 
degraded in the germ-free animal, presumably 
due to lack of a ;8-glucosidase of bacterial ori- 
gin. Therefore, the cycasin ingested by a germ- 
free rat could be recovered nearly quantita- 
tively in the excreta. The metabolic inability of 
the germ-free rat to break down cycasin to its 
aglycone is almost certainly responsible for the 
absence of toxic manifestations in germ-free 
animals receiving cycasin. It is of particular 
significance, therefore, that the recently 
synthesized aglycone given to us by Dr. Matsu- 
moto is highly toxic in germ-free rats. (Dr. 
Spatz and Mr. McDaniel [LNE-NIAMD].) 

Cycasin and Its Aglycone, Methylazoxymethanol 
(MAM), Employing Bacteria 

The effects of cycasin and MAM on bacteria 
are being studied in the hope of discovering 
mechanisms for the hepatotoxicity and carcino- 
genesis of these compounds in animals. Prelimi- 
nary experiments have shown that relatively 
high concentrations (about 20 mM) of MAM 
are required to inhibit the division of Salmo- 
nella typhimimum-. MAM also acts as a muta- 



174 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



gen and causes reversion to wild type of a group 
of histidine requiring Salmonella mutants which 
were also caused to revert by the alkylating 
agent ^-propiolactone. The role of bacteria in 
deglucosylating cycasin is also being investi- 
gated, and an assay for bacterial j8-glucosidase 
using cycasin as a substrate is being developed. 
(Dr. Smith.) 

LABORATORY OF CHEMICAL BIOLOGY 

The Laboratory of Chemical Biology contin- 
ues to have, as its main research goals, prob- 
lems relating to structure-function relation- 
ships in proteins, to the genetic control of 
biosynthesis, and to three-dimensional struc- 
ture. 

Structure-function Relationships in Proteins 

Continuing studies on the amino acid se- 
quences of the extracellular nuclease of Staph- 
aureus have resulted in a nearly complete pic- 
ture of the covalent structure of this protein. 
There remains only a series of relatively rou- 
tine stepwise degradations of small fragments 
to permit an unambiguous ordering of the 
amino acid residues. This protein which con- 
tains approximately 120 amino acids is simul- 
taneously being examined for its physical and 
catalytic properties. It has been shown by opti- 
cal rotary measurements that the native poly- 
peptide contains approximately 50% helix. 
Crystals of the enzyme have been prepared by 
Dr. Hazen and Dr. Cotton at MIT, and unit cell 
dimensions and the nature of the space group- 
ing are now being established. Comparison has 
been made of the covalent structures of the en- 
zymes from two strains of staph. They appear 
to be nearly identical. Upon completion of the 
present chemical and physical work it is 
planned to place heavy emphasis on the nature 
of the active center and on the geometric ar- 
rangement of the chain in three-dimensions. 
(Drs. Taniuchi, Anfinsen, Heins, and Suriano.) 

Purification of the S-RNA synthetase for ty- 
rosine and alanine from E. colt cells is nearly 
completed. Experiments are now in progress to 
determine whether these two enzymes of simi- 
lar function exhibit closely similar chemical 
and physical properties. These studies will test 



the hypothesis that the whole array of synthe- 
tases may be variations on the same structural 
theme. (Dr. Herzig.) 

Since the most effective method, in principle, 
for the study of structural function relation- 
ships would be the direct organic chemical mod- 
ification of amino acid sequence and chain 
folding, we have continued our collaborative 
work with Dr. B. Wildi of the Monsanto Chemi- 
cal Company. Previous studies established that 
protein amino groups could be blocked by tri- 
fluoroacetyl groups. We have now developed a 
useful reversible blocking agent for carboxyl 
groups as well, namely N-chloromethyl pthali- 
mide. Bovine pancreatic ribonuclease and the 
staph nuclease referred to above can be com- 
pletely masked with this reagent and enzymat- 
ic activity can then be regenerated by treat- 
ment of the protein derivative at 0° with 1 M 
piperidine. This situation now permits a syste- 
matic study of the trypsin digestion of the 
blocked protein to cleave arginyl bonds specifi- 
cally. It is planned to prepare and separate 
large fragments using such an approach and 
then to reassemble these fragments with and 
without prior treatment to shorten or lengthen 
or otherwise modify individual fragments. 
Since, as is discussed below, the folding of the 
polypeptide chain into the specific, catalytically 
active three-dimensional structure appears to 
be spontaneous, these studies may eventually 
permit a partial sjoithesis of an enzyme of 
moderate size. (Dr. Wildi, Dr. Goldberger, and 
Dr. Anfinsen.) 

Formation of Tertiary Structure of Proteins 
from the Primary Sequence 

Several further examples of the spontaneous 
refolding of reduced polypeptide chains in the 
native form containing the proper pairing of 
half-cystine residues have been studied. Taka- 
amylase and soy bean trypsin inhibitor can be 
regenerated from the random uncrosslinked 
chain in essentially quantitative yields. This 
process is catalyzed by an enzyme which we 
have now prepared in pure form from extracts 
of acetone powders of beef liver microsomes. 
The purest preparation causes the complete 
reactivation of a soy bean trypsin inhibitor 
derivative containing randomly paired half- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



175 



cystine residues in less than 5 minutes in a mo- 
lar ratio of enzyme to protein substrate of less 
than 1 to 200. It is now fairly clear that the 
enzyme acts as a protein disulfide isomerase 
and converts incorrect to correct SS pairs. The 
purified enzyme is being examined chemically. 
Its availability now permits a direct study of 
the mechanism of action. The enzyme is being 
used as a probe into the thermodynamic stabil- 
ity of various proteins and protein derivatives. 
It has been shown, for example, that native in- 
sulin is rapidly converted to a high molecular 
weight net work of disulfide bonded chains 
which precipitate from solution. These experi- 
ments suggest that the tertiary structure of 
insulin is thermodynamically unstable and that 
the protein may not be synthesized by the 
simple combination of the two preformed 
chains through SS bonds. (Dr. Givol, Dr. Gold- 
berger. Dr. Anfinsen, and Dr. De Lorenzo.) 

Since the enzyme mentioned catalyzes disul- 
fide interchange, we are using it to examine 
the equilibrium concentrations formed during 
the simultaneous oxidation of unlike peptides 
containing SH groups. Such equilibria, which 
are very difficult to obtain in uncatalyzed sys- 
tems, may yield information about tiie nature 
of interactions between amino acid side chains 
which favor the formation of specific disulfide 
bonds in proteins. (Dr. Fuchs, Dr. De Lorenzo, 
and Dr. Anfinsen) 

Control Mechanisms in Protein Biosynthesis 

The genetic control of protein biosynthesis is 
being studied in two bacterial systems, the 
synthesis of j8-galactosidase in E. coli and the 
formation of enzymes of Salmonella typhimu- 
rium which are concerned with the formation 
of histidine. Efforts on the first of these proj- 
ects have been concerned mainly with the pu- 
rification of j8-galactosidase and the study of its 
fundamental chemical and physical character- 
istics. This protein has been shown to have a 
molecular weight of 540,000 and to be com- 
posed of four probably identical subunits, 
which in turn appears to consist of several dif- 
ferent smaller polypeptide units having molec- 
ular weights on the order of 40-50,000. Stud- 
ies are now in progress to establish the degree 
of diflference between these ultimate subunits. 



These studies are essential for a proper un- 
derstanding of the consequences of genetic mu- 
tation in the portion of genome which consti- 
tutes the so-called Lac operon. This operon is 
visualized at present by most workers in the 
field as a series of related structural genes con- 
trolling the synthesis of enzymes involved in 
the uptake and metabolism of j8-galactosides. 
The operon is thought to include a genetic locus 
which produces a specific repressor substance 
designed to combine with and inactivate a sec- 
ond locus termed the operator region. Synthesis 
of messenger RNA corresponding to the struc- 
tural genes in the operon can only occur 
when certain inducers are added to the bacte- 
rial system that can presumably combine with 
the repressor and thus free the operator to ini- 
tiate transcription. It had not been established 
whether the operator is part of the first struc- 
tural gene in the series or a completely sepa- 
rate locus not connected with poly-peptide se- 
quence determination. We have now shown 
fairly convincingly that the j8-galactosidases 
formed by strains of cells both containing, and 
lacking, and effective operator region, are 
chemically identical. Galactosidases from cells 
with an intact operative region and from cells 
possessing a large deletion in this region have 
been found to show the same physical proper- 
ties and to exhibit identical catalytic activities. 
Their covalent structures also are identical on 
the basis of amino acid analysis, peptide map- 
ping, and the variety of other chemical treat- 
ment. 

We are now preparing galactosidase from a 
number of other genetic strains of E. coli and 
propose to examine the consequences of muta- 
tions of various kinds in the structural genes 
themselves. These mutants will include the so- 
called polarity mutants and their revertants. 

The second bacterial system under study, 
namely that for histidine biosynthesis in Salmo- 
nella, has been carefully characterized with 
respect to the time of appearance of various en- 
zymes in the histidine operon after derepres- 
sion of the synthetic sequence. It has been 
shown that the changes in specific activities 
after derepression, of five of the ten enzymes in 
the operon series follow a definite pattern: the 
enzymes encoded into the operon nearest the 



176 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



operator region are the first to show a rise. 
Those enzymes encoded progressively farther 
from the operator region do not begin to show 
an increase in specific activity until consider- 
ably later. The large body of data accumulated 
has been analyzed by computer methods and 
the exact time of appearance of a rise in activ- 
ity for each enzyme has thus been determined 
with some precision. Pulse-labelling experi- 
ments are now being initiated in an effort to 
examine the question of whether a single large 
messenger RNA molecule is synthesized for the 
entire operon or whether the series of individ- 
ual messenger RNA substances are formed 
and released for each of the individual enzymes 
involved. The basis for the sequentiality may 
be either a sequential formation and degrada- 
tion of ten separate messenger-RNA molecules 
or, alternatively, the formation and non-forma- 
tion of a single huge messenger-RNA molecule 
that can be "read" by the protein synthesizing 
system starting only at one end and proceeding 
only in one direction. 

These studies are also now concerned with 
the biosjTithesis of enzymes in the histidine 
operon in strains containing a large deletion 
which eliminates information for all but the 
first or second gene at each end of the operon. 
An analysis of the time characteristics men- 
tioned above should give some information 
about whether deletion mutants are actually 
missing a stretch of DNA or whether the appar- 
ently deleted region indicated by genetic tests 
could conceivably be a replacement of preexist- 
ing DNA with "nonsense" DNA which is not 
translated in the polypeptide structure. (Dr. G. 
Craven and Dr. E. Steers.) 

Proteins and Human Heredity Disease 

As part of a continued study on the relation- 
ship between protein sequence and conforma- 
tion extensive statistical correlation was car- 
ried out on the basis of data from the large 
literature on mutational and species variations 
in protein structure. The results confirm the 
hypothesis that changes in amino acid sequence 
are only permissible when they permit the for- 
mation of the same three dimensional struc- 
ture. 



In preparation for planned chemical study, 
the genetics of congenital erythropoietic por- 
phyria was analyzed from all of the existing 
pedigrees in the literature. All data were found 
to be compatible with the autosomal recessive 
mode of inheritance. (Dr. Epstein.) 

LABORATORY OF BIOCHEMICAL 
PHARMACOLOGY 

Control Mechanisms for Mammalian Enzymes 

The work in this Laboratory has shown for 
the first time that enzyme levels in animal tis- 
sues are controlled by changes in both enzyme 
synthesis and enzyme degradation. For ex- 
ample, glucocorticoid administration causes an 
increase in tryptophan pyrrolase levels by in- 
creasing the rate of synthesis, while trypto- 
phan administration causes a comparable 
increase in tryptophan pyrrolase levels by in- 
hibiting breakdown of the enzyme. Evidence for 
these conclusions has been obtained by a com- 
bination of kinetic, isotopic, and immunologic 
technics. Subsequent studies on the stability of 
tryptophan pyrrolase have demonstrated that 
the process of enzyme degradation in the intact 
animal or tissue slices is very complex and can- 
not be exactly reproduced in broken cell prepa- 
rations or in metabolically-inhibited tissues. 

Similar kinetic studies on the increase in en- 
zyme levels after hormonal administration 
were carried out with other enzymes. Some 
gave rapid responses, others slow responses. 
Careful evaluation, however, revealed that this 
did not represent specificity of response, but 
rather reflected differences in the half-lives of 
the enzymes concerned, i.e., proteins that turn 
over rapidly show a faster response. This is of 
considerable general pharmacologic impor- 
tance. (R. Schimke, C. Berlin, L. Grossbard.) 

Another enzymatic system that offers prom- 
ise for studies of enzyme control mechanisms 
is hexokinase. Four separate types of hexokin- 
ase can be found in rat tissue. Each type has 
different kinetic, electrophoretic, and stability 
properties, as well as different tissue distribu- 
tions. (H. Katzen and R. Schimke.) 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



177 



Microbial Physiology 

ACTINOMYCIN Action in E. coli. Because 
the drug, actinomycin, inhibits the synthesis of 
RNA, it has been of great value in studies of 
protein synthesis, RNA metabolism, and meta- 
bolic control mechanisms. This drug could not 
be used in studies with E. coli, because this or- 
ganism is normally impermeable, and therefore 
insensitive, to actinomycin. It now has been 
shown that this permeability barrier can be 
temporarily removed by briefly treating E. coli 
with the chelating agent, EDTA. The bacteria 
become permeable not only to actinomycin, but 
to a variety of compounds (e.g., o-nitro- 
phenyl-/?- D-galactoside, carbamylphosphate) 
which normally cannot enter E. coli. This 
EDTA treatment does not cause any decrease 
in viability or in the ability to synthesize pro- 
teins or nucleic acids. (L. Leive) 

The use of the EDTA treatment for sensitiz- 
ing E. coli to actinomycin should be extremely 
fruitful since so much is known about the genet- 
ics and enzymatic processes of E. coli. Several 
problems have already been undertaken. For 
instance, the degradation of messenger RNA 
and the assembly of ribosomes from ribosomal 
RNA and protein is being studied in actinomy- 
cin-inhibited E. coli. (L. Leive) 

Other studies have concerned the effect of 
actinomycin on the replication of bacteriophage 
T4 in E. coli. Somewhat surprisingly, ex- 
tremely low concentrations of actinomycin pre- 
vent the formation of mature phage, even 
though this concentration of actinomycin has 
no effect on RNA, DNA, or protein biosynthe- 
sis. These results suggest that these low 
concentrations of actinomycin inhibit the 
packaging of the phage DNA rather than the 
synthesis of messenger. (D. Korn, L. Leive, J. 
Protass) 

Bactriophage (Lysogenic Induction). 
Studies with the lysogenic strain, E. coli 
K12 (A), have shown: (1) Superinfecting 
phage are excluded from E. coli K12 X only if 
prophage DNA synthesis can occur. (2) It is 
known that E. coli K12 A, infected by Xind", 



cannot be induced; they are "repressed." If the 
bacteria are first induced such cells rapidly be- 
come resistant to this repression by Aind". It 
has now been shown that Aind- does get into 
such cells, but is prevented from acting by 
some intracellular change occurring immedi- 
ately after induction. (3) In experiments in 
which lysogenic induction was caused by mito- 
mycin C, uracil has to be present during the 
induction period to allow prophage DNA repli- 
cation to occur. In the absence of uracil, pro- 
phage loses the ability to replicate, but can still 
direct the synthesis of the phage-specific exo- 
nuclease. (D. Korn, J. Protass) 

Ribosomes. A new method has been devel- 
loped for the preparation of highly-purified ri- 
bosomes from E. coli. These ribosomes have 
considerably higher RNA/protein ratios than 
the preparations currently in use. (A. Furano) 
Dye-binding studies have been carried out on 
ribosomes, and indicate that most of the RNA 
in ribosomes is in a coil conformation. (A. Fu- 
rano and D. Bradley) 

Hfr Transfer. Studies have been carried out 
on the mechanism of chromosome transfer in 
mating of E. coli K12. Preliminary experi- 
ments indicate that concomitant DNA synthe- 
sis is not required for chromosome transfer. 
(G. Crowley) 

Spermine, Spermidine and Other Amines 

This laboratory has been interested for 
many years in the metabolism of various bio- 
logic amines, particularly histamine and various 
polyamines. During the past year special atten- 
tion has been directed toward the metabolism 
of spermine, spermidine, and 1,4-diaminobu- 
tane (putrescine) . These are naturally-occur- 
ring amines, which, in recent years have at- 
tracted special interest because of their high 
affinity for nucleic acids, phospholipids, and 
other acidic components of the cell. 

(1) Further studies have been carried out 
on the Serratia enzyme that carried out the fol- 
lowing oxidation: 



NHj(GHj)iNH(CH!)3NH2 + Oj 
Spermidine 



-> NH3(CH,)3NHj + HjC 



N 



-CHj 
CH + HjOj 



178 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



This enzyme, as opposed to other amine oxi- 
dases, will not react directly with molecular ox- 
ygen, but required the addition of an electron 
carrier, such as phenazine methosulfate. Acid 
ammonium sulfate treatment results in disso- 
ciation of the enzyme from its cofactor ; activity 
can be restored by addition of flavin adenine 
dinucleotide. (A. Campello, C. Tabor and H. 
Tabor) . 

(2) Extensive studies have been carried out 
on the monoamine oxidase that has been pu- 
rified 5,000-fold from human plasma. Kinetic 
studies have demonstrated that the unionized 
amines are the effective substrate form for this 
enzyme. Clinical studies have shown that ele- 
vated enzyme levels are found in some cases of 
chronic congestive failure and of chronic liver 
disease. (C. McEwen) 

(3) A monoamine oxidase has been purified 
650-fold from rabbit sera, and extensively stud- 
ied. In contrast to the human serum amine ox- 
idase, the protonated amines are the effective 
substrates for this enzyme. (C. McEwen) 

(4) Active transport systems have been dem- 
onstrated in E. colt for 1,4-diaminobutane, 
spermidine, and spermine. (C. Tabor and H. 
Tabor) 

(5) The metabolism of 1,4-diaminobutane 
and spermidine has been investigated further 
in E. coll and B. subtilis. Methods have been 
developed for the isolation of amino acid-sper- 
midine conjugates from E. coli. (C. Tabor, H. 
Tabor, L. deMeis, D. Savitz) 

(6) Studies have been continued with beef 
plasma amine oxidase that carry out the reac- 
tion: 

NH,(CH2),NH(CH2)3NH3 + 1 0, + 1 H,0 ^ 

NH2(CH2),NHCH2CH.CHO + 1 NH3 + H^O^ 
Spermidine 

Chemical studies on the reaction product show 
that, upon heating, the dialdehyde produceis 
degraded to 1,4-diaminobutane and acrolein. 
(C. Tabor and H. Tabor) 

(7) Derivatives of imidazoleacetaldehyde 
have been synthesized. This is the postulated 
product of the diamine oxidase catalyzed oxida- 
tion of histamine. (H. Bauer) 



Experimental Bums 

Experiments with germ-free mice have 
shown that infection, particularly with E. coli, 
plays an important role in mortality after 
burns. Because of these observations endotoxin 
toxicity was studied in burned and in normal 
mice; endotoxin was about 1,000 times more 
lethal in burned mice than in normal controls. 
Mice tolerant to a lethal dose of endotoxin dem- 
onstrate significantly lower shock mortality 
after burns than non-tolerant mice. (K. Mark- 
ley) 

Leprosy 

Studies have been continued on improving 
the growth of Mycobacterium leprae murium 
in cultures of mouse peritoneal macrophages. 
Best growth was observed when the mediimi 
was supplemented with both ferric nitrate and 
a liver fraction. Under these conditions the 
generation time of M. leprae m,urium was 
7-8.5 days. Studies are in progress to apply 
these methods to the culture of M. leprae (the 
human strain). Chemotherapeutic studies on 
murine leprosy in vivo have also been contin- 
ued. (Y. Chang) 

Sulfur-containing Compounds 

The enzyme glutathione reductase possesses 
one equivalent of flavin adenine dinucleotide 
(FAD) per mole and one -SH group appears 
upon reduction of the molecule with TPNH. It 
had been postulated that in the oxidized form 
of the enzyme the sulfur atom of this -SH 
group is covalently bound to FAD by a TPNH- 
reducible bond. Such a bond is pictured as be- 
ing repetitively opened and closed in the course 
of the catalytic activity of the enzyme. This 
bond was also thought to be responsible for the 
inability of FAD to dissociate from the apoen- 
zyme without reduction. Although this may be 
true, it has now been shown that a second redu- 
cible bond, distinct from the one discussed 
here, is also involved in the binding of the 
coenzyme. (R. Colman, S. Black and S. 
Hopper) 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



179 



Effect of Fasting on Fatty-changes in Mice 

The effect of fasting and of exercise was 
tested on organ weight, water and neutral fat 
content and histology of various organs. (S. 
Webster and B. Highman) 

LABORATORY OF BIOCHEMISTRY AND 
METABOLISM 

Carbohydrate Metabolism 

Reactions Involving Carbohydrate Poly- 
mers. An unusual amino sugar nucleotide, 
TDP-N-acetyl-3-amino-3,6-dideoxy hexose, was 
synthesized by extracts of Xanthomonas cam- 
pestris. The acetylated monosaccharide was 
then isolated in crystalline form, and its struc- 
ture tentatively determined as N-acetyl-3- 
amino-3,6-dideoxy-D-gaIactose. This sugar has 
been identified as a constituent of a toxic, 
phenol-soluble lipopolysaccharide obtained from 
the same organism. (Drs. G. Ashwell and J. 
Hickman) 

Studies have been carried out which, it is an- 
ticipated, will provide meaningful insight into 
the complicated reaction mechanism of deoxy 
sugar formation. These involve study of the 
utilization of TDP-glucose specifically labelled 
with tritium on carbon 3. Degradation studies 
were carried out on an enzymatic reaction prod- 
uct, L-rhamnose (a 6-deoxy sugar) and the 
reaction intermediate, TDP-4 keto-6-deoxy glu- 
cose. (Dr. 0. Gabriel) 

A galactose polysaccharide of snails, called 
galactogen, contains both Z>-galactose and the 
rare sugar, L-galactose. The biosynthesis of 
this polymer has been demonstrated utilizing 
UDP-D-galactose-C" as a galactosyl donor. 
(Dr. E. Goudsmit) 

The mechanism of glycosylation of plasma 
glycoprotein has been studied. Rat liver micro- 
somes contain the enzyme required to attach N- 
acetyl neuraminic acid to a variety of glycopro- 
tein acceptors and also contain an endogenous 
acceptor. Evidence was found that in mucopoly- 
saccharide formation the synthesis of protein- 
is synchronized with formation of the polysac- 
charide moiety. (Drs. E. Neufeld, P. J. O'Brien 
and C. C. Levy) 



Reactions Involving Small Carbohydrate 
Molecules. A number of investigations have 
been concerned with the metabolic control of 
biosynthetic pathways by feedback inhibition, 
and some interesting and intricate mechanisms 
were discovered. These include the following: 

1. Certain bacteria contain fucose but not 
mannose in their polysaccharides, while others 
do contain mannose, or mannose plus fucose. 
The biosynthetic pathway leading to these sug- 
ars involves formation first of GDP-mannose, 
then of GDP-fucose. Their synthesis was inde- 
pendently controlled by inhibition of the pyro- 
phosphorylases by these coenzymes. Similarly, 
CDP-glucose pyrophosphorylase, an enzyme 
which catalyzes the first step in CDP-paratose 
biosynthesis, was inhibited by CDP-paratose. 
(Drs. R. Kornfeld, R. Mayer and V. Ginsburg) 

2. In rat liver, formation of UDPG (the glu- 
cosyl donor in glycogen biosynthesis) was inhib- 
ited by 5'-AMP and by UDPG. (Dr. S. Korn- 
feld) Also in rat liver, the feedback control of 
sugar nucleotide biosynthesis was investigated. 
For example, it was found that UDP-N-acetyl 
glucosamine inhibited L-glutamine D-fructose- 
6-P transaminase, the first enzyme unique to 
its biosynthesis. Similarly, the formation of N- 
acetyl mannosamine by UDP-N-acetyl glucosa- 
mine 2-epimerase is inhibited by the end prod- 
uct CMP-U-acetyl neuraminic acid. This feed- 
back inhibition was also demonstrated in vivo. 
UDP-glucose dehydrogenase of liver, peas and 
cartilage is strongly inhibited by UDP-xylose. 
This nucleotide is known to be the end product 
of the UDP-glucose ^ UDP-glucuronic path- 
way in peas. (Drs. E. F. Neufeld, P. J. O'Brien, 
S. Kornfeld and R. Kornfeld) 

The general principle emerging from these 
and many other studies in the literature is that 
the end product of a biosynthetic pathway con- 
trols its rate of synthesis by specific inhibition 
of an enzyme concerned with an early step in 
the biosynthesis. 

An enzyme fraction from the "high-speed su- 
pernatant" of rat testis catalyzed the conver- 
sion of glucose-6-phosphate-C" to the cyclic 
compound, inositol. The system requires DPN 
and Mg". (Dr. F. Eisenberg, Jr.) 



180 



ANNUAL REVIEW OF INTKAMURAL RESEARCH 



A new enzyme activity, dTDP-N-acetyl-D- 
glucosamine pyrophosphorylase, was shown in 
extracts of hog gastric mucosa. (Drs. R. Korn- 
feld, S. Kornfeld and V. Ginsburg) 

Intermediary MetaboIiBm of Hydrogen 

These investigations are concerned with a 
rather extreme case of biological specificity — 
the metabolic behavior of hydrogen atoms 
whose location in the molecule is slightly differ- 
ent, for example, the metabolic behavior of the 
two enantiomorphic carbinol hydrogens on car- 
bon 1 of fructose-6-phosphate in mammalian 
liver. For such studies the hydrogens are spe- 
cifically labeled with tritium (H^). The initial 
studies have sho^vn that in mammalian liver 
tissue the intracellular synthesis of the two 
distinct H^-labeled carbinol hydrogens of fruc- 
tose-6-phosphate can be effected. One of these 
is formed on incubation with ribose-1-H^ the 
other on incubation with mannose-1-H^. The 
ribose label eventually appears in position 1 of 
glucose-6-phosphate whereas mannose-1-H^ 
gives rise to fructose-6-phosphate so labeled 
that on isomerization to glucose-6-phosphate 
the H^ appears as triton or in position 2 of glu- 
cose-6-phosphate. (Drs. B. Bloom and J. Mil- 
let) 

Hormones 

An investigation is under way to study, at a 
molecular level, the mechanisms by which hor- 
mones regulate morphological and biochemical 
differentiation and growth of mammalian tis- 
sues. Twelve-day pregnant mouse mammary 
p-land explants are cultured in a chemically- 
defined medium using the floating lens paper 
technique. A combination of three exogenous 
Hormones, insulin, prolactin and hydrocorti- 
sone, was most effective in stimulating secre- 
tion of proteinaceous material, in casein bio- 
synthesis and in stimulation of P'^ uptake into 
ribosome-associated RNA. Proliferation of 
epithelial cells was about as good in the ab- 
sence of hydrocortisone. Further analysis of 
the effect of individual hormones on this in 
vitro differentiation system is under way. 
(Drs. Y. Topper, W. Juergens and F. Stock- 
dale) 



A purified enzyme from mammalian liver, 
glutathione-insulin transhydrogenase, cata- 
lyzes the reductive cleavage of insulin with 
glutathione. Recently it has been found to be 
active with a number of modified form of insu- 
lin. Further, the enzyme stimulated the rate, 
but not the extent, of formation of insulin-like 
protein from reduced precursors during air ox- 
idation. Thus, it is possible that the enzyme is 
involved in generation of action insulin from 
its polypeptide chain precursors. The enzyme 
was also active in accelerating the rate of 
regeneration of reduced pancreatic RNase 
during the course of air oxidation. (Drs. F. 
Tietze and H. M. Katzen) 

Nucleic Acids and Other Polynucleotides 

Structure and Function of S-RNA. 
Periodate oxidation of the ribose moiety of 
the terminal adenosine residue of S-RNA de- 
stroys its acceptor activity. Such S-RNA com- 
petitively inhibits the enzymatic aminoacyla- 
tion of native S-RNA, and the effect is specific. 
Thus, a periodate treated fraction originally 
rich in valine activity gave good inhibition of 
the aminoacylation of valyl-s-RNA but not of 
tyrosyl-s-RNA and vice versa. Partial diges- 
tion of S-RNA with snake venom phosphodies- 
terase also yielded an inhibitory product. In 
other work, an immunochemical method was 
devised for purification of S-RNA. (Drs. G. 
Schwartz, J. Torres and M. Kern) 

A new, sulfur-containing nucleotide was dis- 
covered in S-RNA from E. coli. It is 4-thiouri- 
dine phosphate, a material never before found 
in nature. It has been purified and identified. 
Work is under way concerning its role in sec- 
ondary structure, and its route of biosynthe- 
sis. (Dr. M. Lipsett) 

The antibiotics, mitomycin C and porfiromy- 
cin, were found to alkylate DNA, ribosomal 
RNA and S-RNA. Presumably this is part of 
their mechanism of biological action. From 
S-RNA alkylated with mitomycin, both a mo- 
noguanyl and diguanyl derivative were ob- 
tained. (Drs. M. Lipsett and A. Weissbach) 

Enzymes Active with RNA. A highly 
purified, potassium-activated phosphodiester- 
ase of E. coli was found to be extremely spe- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



181 



cific for single-stranded polyribonucleotides. It 
is exquisitely sensitive to secondary structure, 
being unable to hydrolyze known helical pol- 
ymers as (poly A + 2 poly U). The enzyme 
was used to follow the melting out of certain 
helix-random coil systems. It is possible that 
this diesterase is responsible for the break- 
down of messenger RNA, especially since it is 
40 times more abundant than polynucleotide 
phosphorylase, and 3 times more abundant 
than polynucleotide phosphorylase, and 3 times 
more abundant than ribosomal RNase, in ex- 
tracts of E. coli. A general method for the 
synthesis of trinucleoside diphosphates with 
polynucleotide phosphorylase was developed. 
This is very useful for studies of the genetic 
code. The sensitivity of both polynucleotide 
phosphorylase and phosphodiesterase to second- 
ary structure was further established by com- 
paring the degradation of free poly U and pol- 
ymer bound in the complex (2 moles poly 
U + one mole 2,6-diaminopurine riboside). 
(Drs. M. F. Singer, F. Howard, P. Leder and 
R. Brimacombe) 

Biochemistry op Lysogeny in E. coli 
K12. Lysogenic cells {E. coli K12A.) can be in- 
duced to form vegetative phage with the anti- 
biotic, C"-porfiromycin, and the A phage thus 
produced was found to contain the antibiotic. 
In other studies, it was found that superinfec- 
tion of such immune cells with the homologous 
phage A results in no synthesis of an early 
phage enzyme, A exonuclease. This indicates 
that repression of the superinfecting phage 
genome occurs before the stage of early en- 
zyme synthesis, probably at the level of tran- 
scription of the DNA to form messenger RNA. 
The mutant phage A^ir is able to overcome the 
immunity of E. coli K12A and produce normal 
yields of phage in these lysogenic cells. How- 
ever, the Avir mutant is still partially re- 
pressed in lysogenic cells since its ability to 
produce the A-exonuclease is repressed about 
two-thirds when compared to Avi,. infection of 
nonlysogenic cells. This indicates that sensitiv- 
ity to repression in lysogenic cells may be par- 
tial or complete. (Drs. A. Weissbach and A. Li- 
sio and Mr. W. E. Pricer, Jr.) 



Enzyme Induction in a Broken Cell Preparation 

Partial success has been achieved in prepar- 
ing a system of digitonin-lysed, penicillin 
spheroplasts of high synthetic activity, in 
which newly synthesized /3-galactosidase repre- 
sents as much as 2-3% of the initial protein. 
The final preparation is not free of cells or 
spheroplasts. However, by means of visual and 
viable counting techniques it has been estab- 
lished that the cell count decreases in the 
course of incubation. Equally encouraging is 
the demonstration that E. coli, particularly 
after having been exposed to digitonin, form 
little or no jS-galactosidase under the experi- 
mental conditions employed. Thus it is unlikely 
that the observed enzyme synthesis was due to 
contamination by intact cells. (Drs. I. G. Leder 
and J. C. Rabinowitz) 

Enzymatic Utilization of Model Compounds 

Several diverse enzyme systems are under 
investigation as part of a continuing study of 
the reactivity of a variety of chemical group- 
ings in enzyme-catalyzed reactions. As one ex- 
ample, the enzyme responsible for the synthesis 
of niacin, quinolinate phosphoribosyl transfer- 
ase, has been isolated in crystalline form. It cat- 
alyzes a reaction between quinolinic acid and 
5'phosphoribosyl-pyrophosphate to give nico- 
tinic acid mononucleotide, CO2 and pyrophos- 
phate. Free nicotinic acid is not an intermedi- 
ate. Current experiments are concerned with 
the confirmation of exchange reactions between 
enzyme and substrate which are predicted by 
kinetic analysis of the process. As another ex- 
ample, two enzyme systems for tartaric acid 
utilization are being investigated with regard 
to the enzymes and intermediates which are in- 
volved. One of these converts L ( -F ) -tartaric 
acid to oxalacetic acid, and the other converts 
meso-tartaric acid successively to dihydroxyfu- 
maric acid and Z)-glyceric acid. (Drs. W. Ja- 
koby, P. Packman and L. Kohn) 

Release of Surface Enzymes from E. coli by Os- 
motic Shock 

E. coli cells were subjected to osmotic shock 
by suspension in a hypertonic solution of su- 
crose with 3 X 10-^ M EDTA, followed by sus- 



182 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



pension in cold water or 10"^ M MgCL. The 
sudden osmotic transition caused 3% of the 
cellular protein to be ejected into the medium 
within one minute. Most of the acid-soluble 
pool was also lost, and yet the cells remained 
viable. A special set of proteins was released, 
including alkaline phosphatase, 5'-nucleotidase, 
cyclic phosphodiesterase, acid phosphatase and 
RNase. Histochemical evidence (Bruce Wetzel) 
and other experiments suggest that these de- 
gradative enzymes are at or near the cell sur- 
face. These experiments are significant for the 
following reasons: (1) They indicate that de- 
gradative enzymes are somehow segregated in 
the bacterial cell. (2) They provide for biologi- 
cal study viable E. coli lacking these degrada- 
tive enzymes and with increased permeability. 
(3) They provide an easy and effective route 
for purification of certain enzymes. (4) Other 
systems can then be studied in E. coli without 
interference from phosphatases. (Drs. W. 
Brockman, N. Nossal and M. N. Lipsett) 

LABORATORY OF PHYSICAL BIOLOGY 

The past year has been a busy and produc- 
tive one, with about 50 publications, involving 
about 30 intramural authors. As in previous 
years we have enjoyed the stimulation of vis- 
iting colleagues from abroad; this year's crop 
including representatives from Chile, England, 
Israel, Japan, Mexico and Switzerland. 

Present interests of LPB scientists happen 
to fall rather naturally into three main do- 
mains, molecular structure, metabolism, and 
biological energy transduction. In reporting 
highlights of these investigations only the 
names of intramural workers making major 
contributions are given. The numerous addi- 
tional collaborators, past and present, includ- 
ing those outside our Laboratory, are credited 
in the various individual reports. 

Molecular Structure 

The Laboratory of Physical Biology is en- 
gaged in an intensive and diversified investiga- 
tion of the structures of molecules ranging 
from simple inorganic compounds to macro- 
molecular polymers, using all the standard 
spectroscopic tools, a battery of automated de- 
vices for recording and converting spectral 



data into numerical form suitable for calculat- 
ing the corresponding molecular parameters, 
and computer matching with theoretical mod- 
els. When applied to molecules such as hemo- 
globin or polypeptides the biomedical connota- 
tions of such interests are clear. But it perhaps 
needs to be emphasized that the physico-chemi- 
cal parameters of simple molecules, esoteric as 
such data appear, are of possibly even greater 
importance in the long run, since this informa- 
tion is more basic and broadly applicable. 
Further, the types of apparatus needed for 
studying molecular structure are so complex 
and the principles of operation are so well con- 
cealed in "black boxes" that it may be worth- 
while to attempt some slight explanation of the 
enigmatic initials ORD, NMR, and so on. 

When quantized electromagnetic radiation 
passes through a material, some of its energy 
may be absorbed providing there is a very pre- 
cise fit between the kind of energy offered — 
that is, the wavelength (frequency) of the ra- 
diation — and the ability of the absorbing mole- 
cule to receive it. Absorbed energy may cause a 
variety of intramolecular changes: rise of an 
electron to a higher energy level, change of 
axis of spin of electron or proton, reduction or 
increase in rotational or vibrational energy of 
a bond between atoms — all by quantal 
amounts. Hence by scanning molecules of one 
substance with a continuous succession of radia- 
tion wavelengths it is possible to plot an absorp- 
tion spectrum, each peak of which is related to 
a specific structural property or interaction 
of the absorbing molecule. Whether or not 
an absorption peak can in fact be referred back 
to a specific molecular structure usually 
depends on a laborious comparison of spectra 
from a large number of related compounds to- 
gether with computational predictions based on 
thermodynamic and quantum mechanical 
theory. 

In our Laboratory the classical method of in- 
frared spectroscopy, particularly useful for 
measuring molecular vibrational and rotational 
energies, has been used in an important series 
of investigations resulting in a complete "vi- 
brational assignment" for the p-benzoquinone 
molecule and a number of its derivatives 
(Becker, Charney). This means that the 



NATIONAL INSTITUTE OP ARTHRITIS AND METABOLIC DISEASES 



183 



strengths of the two C = bonds, the four 
C— H bonds, the four C— C bonds and the two 
C=:C bonds are precisely known and their in- 
teractions with each other explained. By the 
same methods the electronic distribution pat- 
terns in germanium and tin hydrides, and tri- 
halides of boron, have been investigated, the 
data being used to clarify the theory of force 
fields within these molecules (Levin) . 

Other studies have been carried out using ul- 
traviolet radiation not in simple absorption 
spectroscopy but in the more powerful tech- 
niques of optical rotatory dispersion and circu- 
lar dichroism. These methods are particularly 
adapted to studying "optically active" com- 
pounds (those with mirror-image molecular 
asymmetry) , which are often of special biomed- 
ical importance because one isomer may be 
extremely active physiologically, the other 
completely inactive. The former method 
(ORD) measures the degree of rotation of 
plane-polarized light as a function of wave- 
length; the latter (CD) depends on preferential 
absorption of right or left circularly polarized 
light. Both methods yield information about 
the spatial configuration of atoms in molecules. 
They have been used in studying the stereo- 
chemistry of morphine and codeine derivatives 
(Weiss). Another of our particular interests 
has been the class of conjugated cyclic dienes. 
The basic problem has been to relate the two 
possible configurations of the structure RC = C 
C=CR (that is, the "cisoid" case when the 
molecule is bent at the central C— C bond into 
a V shape, versus the quasi-linear "transoid" 
arrangement when one "arm" is up, the other 
down) to whether the molecular skewing is 
right or left-handed. Study of numerous repre- 
sentative diences has confirmed the relation 
discovered last year that sign of rotational 
effect is related to handedness (Weiss, Ziffer) . 
A comprehensive analysis of the intensity of 
these effects by molecular orbital theory has 
also been completed (Charney) . 

Two other relatively new spectroscopic 
methods, employing magnetic resonance, are 
being widely used in our Laboratory. These 
methods involve subjecting a solution of the 
molecules in question to a strong magnetic 
field, which "freezes" that small fraction of 



the total molecular population that contains 
unpaired electrons or protons whose spin axes 
happen to be parallel to the lines of magnetic 
force. When, now, radiation is passed through 
the molecular aggregation at right angles to 
the magnetic field, some of the oriented parti- 
cules may absorb energy of specific (resonant) 
frequency (wavelength) and "flip" with refer- 
ence to their spin axes. The exciting radiation 
for such electron spin resonance (ESR) is in 
the microwave region, and for proton or nu- 
clear spin resonance (NMR) is in the short- 
wave radio band. ESR has been used to define 
the configuration of the unpaired electrons and 
to account for paramagnetic properties of cer- 
tain halogen compounds of Cr, Va, Mo, W and 
Co (Kon) . NMR was used in a study of binding 
of copper by nucleotides (Becker) ; in an exten- 
sive continuing study of DDT analogs and de- 
rivatives, with a possibility in view of associat- 
ing the toxicity with the lability of a particular 
aliphatic proton (Sharpless) ; and to elucidate 
the structure of a series of important carcino- 
gens, the benzanthracenes (Ziffer) . 

The technique of aligning linear molecules 
with large dipole moments in a strong electrical 
potential and then looking at them with polar- 
ized ultraviolet light, (Kerr technique) is val- 
uable for determining the size and configura- 
tion of polymers. Work of this sort is going 
forward on polypeptides (Yamaoka). In an- 
other unit of our Laboratory evidence is accu- 
mulating that the changes in secondary struc- 
ture of the hemoglobin molecule, brought about 
by a seemingly minor chemical change in one 
of the four polypeptide chains of the globin 
moiety, not only cause the pathological "sick- 
ling" of the erythrocyte but actually favor the 
polymerization of the quasi-tetrahedral hemo- 
globin units into a beaded linear molecular fila- 
ment. Indirect evidence for such aggregation 
from fluorescence microscopy reported last 
year has now been supplemented by showing 
that the sickled cells orient in a magnetic field; 
and by electron microscopy of the extracted he- 
moglobin molecular filaments (Murayama). 
Configurational changes brought about by com- 
plexing mercury with hemoglobin have also 
been studied (Resnik). 

The most important source of information 
about crystal structure of macromolecules is 



184 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



usually X-ray diffraction, but when this fails, 
information can sometimes be deduced from 
electron microscopy. Catalase, for example, a 
heme protein of 225,000 molecular weight, was 
shown as early as 1937 to crystallize in needle, 
rectangular plate and quadrilateral prism hab- 
its, but resisted efforts to derive its crystal 
structure by X-ray diffraction analysis. In our 
Laboratoiy two additional crystal habits (ap- 
proximating the regular octahedron and the 
hexagonal prism) have been prepared, the 
needle habit has been shown to be actually 
clusters of long narrow plates, and crystals of 
any of the four valid habits have been dissolved 
and recrystallized in all of the other habits by 
salt concentration control (Labaw) . 

Metabolism 

At the direct biochemical level a comprehen- 
sive study of nutrition and growth in the green 
flagellated protozoan EugleTia has been com- 
pleted, including development of a defined 
synthetic culture medium, elemental and gross 
chemical analyses and identification of several 
conventional enzymatic pathways of interme- 
diary metabolism (Kempner, Miller) . In other 
work on Protozoa, four protozoan blood para- 
sites were found to differ specifically in their 
abilities to synthesize fatty acids from defined 
substrates (Greenblatt). An interesting new 
approach to bacterial metabolism is suggested 
by the discovery that rates of utilization of va- 
rious substrates and the effects of certain inhib- 
itors during the log phase of growth in E. coli 
are measurable in terms of the amount of elec- 
tric current produced by the cells (Allen) . 

A study is also underway of protein synthe- 
sis during insect development. The particular 
interest here is that many of the structures of 
the immature (larval) stage are completely 
and rapidly made over during the transforma- 
tion into the adult stage. In this study differ- 
ences have been found between the stabilities 
of larval and adult aldolases, an amino acid 
previously unknown in nature (L,d methionine 
sulfoxide) has been discovered, the turnover 
and incorporation rates of lysine and alanine 
have been measured and the changes in titer of 
free amino acids, peptides and proteins in the 
blood during development have been deter- 



mined (Levenbook, Ojeda, Chen) In another 
investigation major enzymatic changes in the 
blood plasma of rats were found in response to 
severe stress by low barometric pressure. It 
was shown that repeated exercise at atmos- 
pheric pressure ameliorated some the damage 
from subsequent exposure to low pressure 
(Altland). 

In the anatomically simple freshwater ani- 
mal Hydra the conditions under which body 
cells transform into germ cells look more and 
more like a complex of quantitative relations 
rather than the various simple triggers that 
have been designated in certain other laborato- 
ries. For example, the sex-inducing action of a 
rise in ambient temperature was shown to be 
quantitatively dependent on both initial and 
final temperature, and to be independent of the 
chemical composition of the microenvironment, 
nutritional state and illuminational regimen 
(Park, Ortmeyer). 

Biological Energy Transduction 

The title of this section is an attempt to char- 
acterize the class of biophysical processes in 
which one type of energy is converted into an- 
other. In the overall view it is easy to see that 
light energy is converted eventually to chemi- 
cal energy in photosynthesis, and that a proc- 
ess descriptively the reverse occurs in biolu- 
minescence. Similarly, chemical energy results 
in mechanical work in the shortening muscle 
and in the active transport of cellular water. 
But as the intimate details of these transduc- 
tions are investigated, more and more subtle 
intercalated steps are revealed and it is doubt- 
ful that any single instance is yet adequately 
understood. Our Laboratory is engaged in a 
broad attack on these problems, including both 
steady-state situations and those that might be 
called biological triggers. 

Perhaps the best known and longest studied 
biological transducer is striated muscle. Elec- 
tron microscopy has shown that the muscle cell 
contains a precisely oriented array of interdig- 
itating protein filaments running parallel to 
the long axis of the cell. The two present main 
theories of muscular shortening provide, re- 
spectively that these filaments shorten by in- 
tramolecular folding and that they slide to- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



185 



gether. Delicate measurements in our Labora- 
tory show that the fine filaments do not change 
length during shortening of the muscle cell, 
thus favoring the second view (Podolsky). Cal- 
cium ion is thought to be the immediate trigger 
of excitation, and this view has been strength- 
ened by localization of intracellular sites of cal- 
cium storage by electron microscopy (Fran- 
zini-Armstrong) , by determining the effective 
concentration range of calcium for excitation 
(Hellam) and by evidence that the calcium is 
liberated intracellularly by depolarization of 
the internal membrane system (sarcoplasmic 
reticulum) which is probably electrically 
coupled to the outer cell membrane (Costan- 
tin). 

The process by which light energy absorbed 
in the retina is converted to an electrical signal 
in optic nerves is under intense investigation 
in many laboratories. In LPB certain steps 
have been studied in the retina of the squid 
(which is particularly favorable for the record- 
ing of the minute electrical currents generated 
by the absorbed light because the receptor cells 
connect directly with single unbranched nerve 
fibers) and it has been possible to show that 
the brief pulses of current that result when 
photons are absorbed do not correspond kineti- 
cally to the rates of transformation of any of 
the known photochemical intermediates. This 
suggests that many steps intervene between 
photon absorption and the eventual change in 
photoreceptor cell membrane permeability, a 
conclusion supported also by theoretical anal- 
ysis of the necessary thermodynamic relations 
between magnitude of membrane current and 
quantum absorption (Hagins). The sophisticat- 
ed computerized "shot noise" analysis used to 
sort periodic electrical signals out of random 
fluctuations in membrane current is also being 
applied to photo emission from luminous bac- 
teria and glowing firefly lanterns (Hagins, 
Hanson) . 

The transfer of light energy absorbed by 
chlorophyll molecules, present in an oriented 
"liquid crystal" state within the layers of the 
chloroplasts of green plants, to the site where 
it is used in the basic photosynthetic reaction, 
has been studied by measuring polarized fluo- 
rescence under the microscope. The results 
point to specific orientation of the pigment 



molecules, the pigment itself being a form of 
chlorophyll absorbing in the far red wave- 
lengths (Olson, Jennings). 

It will be apparent that the leit-motiv in all 
this transduction work is membrane structure, 
cellular or subcellular. Light-absorbing mole- 
cules are oriented on membranes in the outer 
rod segment and in the chloroplast ; changes in 
membrane permeability accompany the pas- 
sage of materials into and out of the cell, the 
generation of the propogated potential of the 
nerve impulse, the spread of excitation within 
muscle cell, and presumably in the transfer of 
nervous excitation to the photocyte. 

In our Laboratory, membrane structure is 
being observed directly by electron microscopy 
(Hagins, Tice, Greenblatt, Peterson) and "liq- 
uid" membranes of strong base ion ex- 
changers are being studied intensively in 
regard to the physico-chemical carrier mecha- 
nism of ion exchange (Sollner, Shean). At the 
same time collodion membranes of controlled 
porosity are being used in quantitative analysis 
of nonelectrolyte and solvent transfer (Gab- 
bay). Another approach is by way of mono- 
molecular films at interfaces. By methods of 
surface chemistry the complex of forces in- 
volved in the specificity and integrity of cer- 
tain such films have been worked out, binding 
of charges has been distinguished from elec- 
trostatic association of electrolytes with the 
monolayer and the effects of certain surface in- 
teractions of steroid hormones have been syste- 
matically elucidated (Gershfeld, Pak). Still an- 
other approach is the finding that similarity in 
ionic radii between arsenate and phosphate al- 
lows the former to substitute for the latter in 
biosynthesis of protozoan cell membrane to 
give a new lipid species, arsenolipids (Sharp- 
less). Such isomorphic replacement, with pre- 
sumably parallel permeability changes, may 
possibly account for the toxicity of arsenic. 

This account of LPB activity reaches full 
circle with the interesting application of nu- 
clear magnetic resonance spectroscopy to intact 
cells of the protozoan Nocardia. NMR is a 
probe in which signal profile is sensitive to 
molecular disorder or asymmetry. Using as a 
control the signal characteristic of disoriented 
lipids when the cell became leaky due to dam- 
age by Ca and Mg ion or by heat. Whether 



186 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



NMR spectroscopy will prove a useful tool for 
studying intact cell membrane behavior re- 
mains to be seen, but these preliminary results 
seem promising. 

In conclusion one is very tem_pted to succumb 
to the invitation of the Director, NIH, to spec- 
ulate about the future, particularly in vievi^ of 
the present prevalence of Planning in the air. 
However, if the history of this elderly Labora- 
tory teaches anything it is that no matter what 
the original intent in bringing scientists to- 
gether or to what professional category the 
scientists nominally belong, no "program" re- 
mains stable for very long. If left free to ex- 
plore, and if not hedged in by artificial organi- 
zational boundaries, independent spirits will 
form a constantly shifting pattern of alliances 
which defies any tidy system of planning, 
reporting or prediction. Such uncertainties, 
however, are more than compensated by the 
f ruitfulness of the interactions in terms of ger- 
minating new departures, introducing concepts 
and techniques from one field into another, and 
efldcient use of available resources. Further- 
more, such free collaboration tends to promote 
an idea-oriented philosophy with emphasis on 
problem-solving, rather than a tool-oriented 
philosophy with emphasis on justifying the ex- 
istence or acquisition of proprietary hardware. 
We feel, therefore, that aside from the minor 
amount of judicious and unobtrusive steering 
that is compatible with research freedom, it is 
better, and more realistic, to simply take pride 
in being part of a productive companionship, 
be thankful for the opportunity to do basic re- 
search, and await with interest the next unpre- 
dicted shift in the harvest. 

LABORATORY OF NUTRITION AND 
ENDOCRINOLOGY 

Guinea Pig Nutrition 

In continuing studies on the quantitative nu- 
tritional requirements of the guinea pig, the 
need for methionine was found to be 0.58% of 
the diet. When adequate cystine (0.24%) was 
present, however, the requirement for methion- 
ine fell to 0.22%. Thus cystine can replace over 
one-half of the methionine requirement of the 
guinea pig. (Dr. M. E. Reid) 



Metabolism and Function of Fat Soluble 
Vitamins and Related Substances 

Analyses of 140 normal adult serums from 
NIH employees revealed an average concentra- 
tion for total tocopherols of 1.06 mg/100 ml, 
with a range of 0.5-2.0. This contrasted with 
an average value of 0.76 mg/100 ml, range 
0.2-1.5, for 125 serums from villagers in East 
Pakistan. Re-evaluation of 40 NIH serums by a 
more precise method showed that a-tocopherol 
comprised 85-90% of the total tocopherols 
with y-tocopherol accounting for the re- 
mainder. 

Further studies of nutritional factors affect- 
ing testis function in rats showed that in the 
absence of dietary polyunsaturated fat a defi- 
ciency of vitamin E does not affect the testis 
for at least 30 weeks (damage ensues in 18 
weeks when polyunsaturated fat is fed) . These 
studies also revealed marked differences from 
the classical symptoms of essential fatty acid 
deficiency, in that rats deprived of linoleic acid 
grew well for 26 weeks and did not have testic- 
ular damage even after 34 weeks. The discrep- 
ancy is thought to be due to improved dietary 
formulation assuring adequate tissue satura- 
tion of all fat vitamins. 

Investigation of the reported reversibility of 
vitamin E-deficiency testis damage in the 
hamster has shown that about one-half of the 
animals had normal restoration of testicular 
tissue while one-half did not. The diflference is 
thought to be due to the extent of vitamin E 
depletion prior to resupplementation with a-to- 
copherol. Even though all hamsters were de- 
pleted for similar long periods, there is an ap- 
parently marked difference in the retention of 
trace amounts of tocopherol by various ani- 
mals. (Dr. J. G. Bieri.) 

Metabolic Adaptation to Protein Deprivation 

When adult male rats were depleted of pro- 
tein and oxidative phosphorylation of the liver 
using succinate as substrate was studied, no dif- 
ference from control rats was found through- 
out the depletion period. However, after 2 
days of protein repletion, oxidative phosphoryl- 
ation fell to a very low level and then returned 
rapidly to a normal level thereafter. The low 
level after 2 days coincident with a rapid infil- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



' 187 



tration of lipid into the liver. When methionine 
or cystine was included in the protein-free ra- 
tion during the depletion, oxidative phosphoryl- 
ation was considerably lower than in controls 
throughout the depletion period. It had been 
shown earlier that inclusion of methionine or 
cystine in the diet during protein depletion 
produced very fatty livers. Thus there appears 
to be somewhat of an inverse relationship be- 
tween accumulation of fat by the liver under 
these conditions and capacity for oxidative 
phosphorylation. 

The ratio of succinate oxidation in an ac- 
tively phosphorylating system ( intact mitochon- 
dria) to that in a non-phosphorylating system 
in which mitochondria were disrupted by cal- 
cium addition was studied in rats depleted of 
protein, to investigate one parameter of mito- 
chondrial membrane permeability. This ratio 
in protein-depleted rats was more than double 
that in control rats. This effect cannot be attri- 
buted to an indirect effect of any one of the 
components added to the in vitro system, such 
as hexokinase, AMP, glucose, KF, Mg+= of glu- 
cose-6-phosphate ; the effect was the same 
whether these components were present individ- 
ually or in various combinations. Thus it 
appears that the permeability of the mitochon- 
drial membrane to succinate is considerably in- 
creased by protein depletion. That the effect is 
not due to uncoupling of oxidative phosphoryl- 
ation in the intact mitochondria is borne out 
by the fact that phosphorylation is normal 
with respect to the amount of oxygen taken up. 

Cytochrome oxidase activity is reduced to 
15% of normal by protein depletion. However, 
cytochrome oxidase concentration (cytochromes 
a -I- as) as measured spectrometrically falls 
to only 50% of normal. Thus it appears 
that while a fairly high concentration of the 
enzyme is still present in protein deficiency, its 
activity is not nearly up to par. This discrep- 
ancy between activity and concentration can 
possibly be explained by a loss of mitochondrial 
phospholipid which occurs in protein defi- 
ciency. It has been shown by others that phos- 
pholipid is necessary for cytochrome oxidase 
activity. However, its presence or absence 
would not affect the spectrophotometric anal- 
ysis for cytochrome oxidase. 



Cytochrome a -I- a^, b, Ci, and c concentra- 
tions (assayed spectrophometrically) are all 
lost to about the same extent during protein de- 
pletion (40-50% of normal). After protein 
repletion it takes at least 8-10 days for the con- 
centrations of these enzymes to return to nor- 
mal. (Dr. J. N. Williams, JR.) 

Lipid Metabolism and Purine-Pyrimidine 
Balance 

Work has continued toward an elucidation of 
the mechanism by which dietary orotic acid in- 
duces accumulation of triglycerides in livers of 
rats. Earlier work has established that lipid ac- 
cumulation results from a severe inhibition in 
hepatic lipoprotein secretion. Furthermore, the 
inhibition of lipoprotein secretion is preceded 
by an elevation in hepatic uridine nucleotide 
concentration, depression in adenine, guanine 
and pyridine nucleotide concentrations, and an 
inhibition in pyridine nucleotide biosynthesis. 
All of these effects of dietary orotic acid are 
prevented and reversed by small amounts of 
dietary adenine. 

More recent findings are summarized here- 
with. Livers were examined histologically and 
with the electron microscope just prior to and 
during fat accumulation produced by orotic 
acid feeding. No abnormalities are seen until 
the third day, at which time small vesicles, pre- 
sumably containing fat, appear between the 
double membranes of the endoplasmic reticu- 
lum. As the fatty liver develops, these vesicles 
increase in size and apparently coalesce, lead- 
ing to the appearance of many round, lipid- 
filled structures throughout the ergastoplasm 
of the cell. (Drs. H. Windmueller and Y. Tan- 
aka) 

The source of the accumulating liver fat was 
studied by examining its fatty acid composi- 
tion. In rats with a high caloric intake, the 
composition was characteristic for newly- 
synthesized fat. When caloric intake was re- 
stricted, the composition was characteristic for 
adipose tissue fat, indicating that the lipid was 
mobilized from peripheral organs before accu- 
mulating in the liver. 

Purine biosynthesis in vivo was measured to 
explain the orotic acid-induced depression of 
purine nucleotides. The incorporation of for- 



188 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



mate-"C and glycerine-2-^*C into the acid-sol- 
uble adenine and guanine of liver was stimu- 
lated 3 to 10-fold by orotic acid. A comparable 
increase vi^as found for the incorporation of 
these precursors into nucleic acid purines of 
liver. Depressed steady-state concentrations of 
purine nucleotides in orotic acid-fed rats are, 
therefore, the result of accelerated catabolism 
or utilization and not due to an inhibition of 
synthesis. 

By measuring the development of hyperlipe- 
mia following an intravenous injection of Tri- 
ton WR-1339, the onset of inhibited hepatic 
lipoprotein section in the rat was found to oc- 
cur after 3 days of feeding orotic acid. Thus, 
inhibited lipoprotein secretion coincides with 
hepatic accumulation although both are pre- 
ceded by progressively diminishing steady- 
state concentrations of lipoprotein in the circu- 
lation. 

Chronic fatty liver and depressed plasma 
lipoprotein concentrations were found in rats 
maintained for 19 months on a purified diet 
containing 1% orotic acid. In addition to being 
fatty, livers of orotic acid-fed rats showed 
adenomatous hyperplasias, fibroadenomas and 
bile duct proliferation. There was no evidence 
of cirrhosis (in collaboration with Dr. G. La- 
queur) . After 19 months of orotic acid feeding, 
a dietary supplement of 0.25% adenine sulfate 
restored liver and plasma lipid concentrations 
to nearly normal levels in 14 days but did not 
reverse the other lesions. (Dr. H. G. Wind- 
mueller) 

Folic Acid 

Several of the forms of folic acid have been 
demonstrated to be co-factors in enzyme sys- 
tems involving the synthesis of thymidine and 
methionine. However, the significance of the 
preponderance of the more complex forms of 
folic acid as they exist in various tissue and 
microorganisms has as yet not been elucidated. 
Our studies in the past year have disclosed that 
the major component (approximately 75%) of 
the folate complex in yeast is a highly conju- 
gated form of 5-methyletrahydrofolic acid. 
More refined isolation and assay procedures 
have been developed. (Dr. J. C. Keresztesy and 
H. A. Bakerman) 



Significant progress has been made in the 
purification and characterization of the proper- 
ties of folic reductase, the key enzyme in the 
conversion of folic acid to the metabolically ac- 
tive coenzyme. The enzyme from chicken liver 
has been purified 8000-fold by a combination of 
gel filtration and column chromatography. This 
preparation appears to be essentially homoge- 
neous and thus was used to confirm the pre- 
viously reported unusually low molecular 
weight of folic reductase, 23,000. A turnover 
number of 350 molecules of dihydrofolic acid 
reduced per minute per molecule of enzyme has 
been obtained for folic reductase which is in 
agreement with drug binding studies. The ob- 
servation that the ratio of activities for the re- 
duction of folic acid vs. dihydrofolic acid is es- 
sentially the same with this 8000-fold purified 
enzyme as compared with the original crude 
preparations is final proof that a single enzyme 
carries out the reduction of both forms of folic 
acid. (Dr. B. T. Kaufman) 

Large-scale Laboratory 

The increased interest of biochemists 
throughout NIH in the structure and synthesis 
of proteins has been reflected in the increased 
use of the fermentation process equipment. On 
the average, four 300-liter fermentations are 
being carried out each full working week. A 
wide variety of non-pathogenic organisms are 
grown under particular cultural conditions as 
requested by the individual investigator. There 
has been an increased use of the other facilities 
of this laboratory for the processing of large 
amounts of natural materials which cannot be 
done with the usual laboratory equipment. 
(D. L. Rogerson, Jr. and Dr. J. C. Keresztesy) 

Experimental Nutrition 

The use of germfree animals continues to 
offer a sophisticated approach to problems in 
nutritional experimentation. The role of the 
microbial flora of the gastrointestinal tract can 
be shown to be either beneficial or detrimental 
to the nutritional status of the host animal. 

SuflScient amounts of the vitamin, folic acid, 
were found to be absorbed directly by rats with 
"normal" bacterial flora to protect such ani- 



NATIONAL INSTITUTE OF AKTHRITIS AND METABOLIC DISEASES 



189 



mals from signs of folic acid deficiency. In con- 
trast, germfree rats develop folic acid defi- 
ciency. 

The studies on the influence of gastrointes- 
tinal flora on amino acid nutrition have shown 
that the administration of antibiotics only par- 
tially reduces the destruction of amino acid 
when compared with germfreeness. Black- 
tongue or kwashiorkor producting diets permit 
much better growth in germfree compared to 
normal rats. 

Experimental dietary liver necrosis studies 
have been extended. More than one type of 
liver lesion was found in rats maintained on 
various deficient diets. In addition to "massive 
necrosis" other lesions which are, as yet, de- 
scribable only as atypical were found. The 
former is more often associated with necro- 
genic diets lacking vitamin E or selenium. The 
latter are more often found as the result of the 
feeding of low protein diets. The addition of 
large amounts of polyunsaturated fatty acids 
reduce the time required to produce both types 
of lesions. While germfree rats are also subject 
to dietary liver necrosis the development of the 
usual massive necrosis is delayed in these ani- 
mals. (Dr. F. S. Daft and E. G. McDaniel.) 

Parathyroid Hormone 

Chemistry. New methods, based on parti- 
tion on Sephadex G-lOO and chromatography 
on carboxymethylcellulose, were devised for 
preparing parathyroid hormone in large quan- 
tities. A variety of physical studies, described 
in previous reports, show that the product is 
better than 90% pure polypeptide, with a molec- 
ular weight of approximately 8500, and 75 
amino acids in length. Time digestions of the 
hormone with exopeptidases have allowed de- 
duction of the amino acid sequences at either 
end of the molecule. The carboxyl-terminal res- 
idue was found to be leucine. Cleavage of the 
molecule with cyanogen bromide yielded one 
major polypeptide, 55 amino acids in length 
from the central portion of the molecule plus 
smaller peptides, 10 amino acids or less in 
length, representing the NHo— and COOH— 
terminal portions of the molecule respectively. 
This was in keeping with the findings from ex- 
opeptidase digestions that, of the 2 methionines 
in the molecule, one is located near either end 



of the polypeptide chain. The hormone was di- 
gested with trypsin and the resulting peptides 
were separated by the "fingerprinting" tech- 
nique. Several of the tryptic peptides have been 
recovered and amino acid analysis completed; 
the COOH — terminal tryptic peptide was ten- 
tatively identified by comparing its amino acid 
composition with results of carboxypeptidase 
digestion of the native molecule. (Dr. G. D. 
Aurbach, in collaboration with Drs. J. T. Potts 
and L. Sherwood of NHI) 

Physiology. Studies were carried out to 
measure the distribution and half -life of par- 
athyroid hormone in the body and to deter- 
mine the physiological factors regulating the 
secretion of the hormone by the parathyroid 
glands. Using pure bovine parathyroid hor- 
mone labeled with "^I it was found that the 
half-life of the hormone in the rat is 22 min- 
utes and the hormone is distributed into a 
space representing 30% of the body weight. 
Further physiological studies based on the im- 
munoassay of the hormone in plasma and pa- 
rathyroid gland perfusates confirmed the 
theory that the rate of secretion of parathyroid 
hormone is governed by the concentration of 
calcium in the blood. Acute hypocalcemia stim- 
ulates whereas hypercalcemia inhibits secre- 
tion of the hormone by the parathyroid glands. 
This study is the first to prove the theory by 
direct measurement of hormone. (Drs. G. D. 
Aurbach and R. Melick, with Drs. L. Sherwood 
and T. Care of NHI) 

Structure-Function Studies and Mech- 
anism of Action of Paratyhroid Hormone. 
Parathyroid hormone stimulates respiration 
and ion transport in mitochondrial systems; it 
is possible that these effects are related to the 
kno\vn physiological actions of the hormone in 
effecting calcium and phosphate transport in 
vivo. The mitochondrial systems afford another 
test parameter for the hormone and its chami- 
cal derivatives; thus structural requirements 
for activity in vitro can be compared with those 
necessary for activity in vivo. The central 
region of the molecule (approx. 55 amino acids 
in length, obtained by cyanogen bromide cleav- 
age) retains considerable immunological and 
in vitro activities but is devoid of biological 
activity as tested in vivo. The COOH — terminal 
region of the polypeptide chain contains a site 



190 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



necessary for highly energetic immunological 
reactivity but is relatively unimportant for bio- 
logical activity. Several amino acids can be 
removed from the NH, — terminal portion of 
the molecule without severely altering any of 
the activities. 

The biochemical mechanism was explored 
whereby parathyroid hormone stimulates res- 
piration of mitochondria in vitro. A sensitive 
test for this effect was developed by measuring 
the hormone-induced degradation of "C-suc- 
cinate to "CO2. Analysis of reaction media 
during hormonal stimulation indicated that pa- 
rathyroid hormone accelerated the rate at 
which DPNH, produced from succinate, was 
reoxidized. It is possible that the parathyroid 
hormone-induced utilization of DPNH is a 
manifestation of hormonal activation of a spe- 
cific ion transport system. (Dr. G. D. Aurbach 
and Dr. J. T. Potts, NHL) 

In collaboration with Dr. Berton Pressman 
of the Johnson Research Foundation, it was 
found that parathyroid hormone in vitro in- 
duces a net transient potassium influx into mi- 
tochondria. This effect is the earliest manifes- 
tation of parathyroid hormone yet found and 
occurs before any change in swelling or respi- 
ration can be detected. (Dr. G. D. Aurbach.) 

Pituitary Hormones 

Bovine TSH. Highly purified bovine TSH 
preparations were further separated by elec- 
trophoresis on slabs of polyacrylamide. The 
separated components were then filtered on 
Gt-100 Sephadex columns. The components of 
lower potency were less retarded on G-lOO 
which indicates unfolding or a larger molecular 
size. Other TSH preparations were fractionat- 
ed by recycling on G-lOO with the separation 
of components with low and high TSH activity. 
Amino acid analyses showed that all fractions 
had a similar composition. This suggests that 
the differences in potency and various physical 
properties of the various TSH fractions are not 
due to differences in primary structure but to 
secondary and tertiary structure to differences 
in charge (de-amidation.) An immunoassay 
procedure for bovine TSH has been developed. 
(Drs. P. G. Condliffe, M. Mochizuke and G. 
Martinoli) 



Murine TSH. Studies have continued on 
TSH in murine pituitary tumors. Previously 
isolated highly potent preparations of the 
mouse tumor TSH have been studied by gel 
electrophoresis using the system described 
above. The active bands have been identified, 
separated and their individual potencies have 
been estimated to be in excess of 100 lU per 
mg. They differ from the bovine TSH compo- 
nents in that they are more acidic and contain 
fewer basic amino acids. This is the highest po- 
tency ever found for TSH gen filtration and 
density gradient centrifugation of the plasma 
from pituitary tumor bearing mice show that 
the hormone in the blood has the same molecu- 
lar weight and electrophoretic mobility as it 
does in the tumors. The TSH in blood can be 
quantitatively recovered and has been purified 
about 100 times by percolation. (Drs. M. Mo- 
chizuki, P. G. Condliffe, and R. W. Bates) 

Bovine Growth Hormone. It was found 
that, as the pH of dilute solutions of the hor- 
mone was lowered from 5 to 2.5, there occurs a 
transition between two distinct configurations 
of the molecule. This transition was studied by 
ultraviolet difference spectroscopy, fluores- 
cence, polarization of fluorescence, viscosity, 
sedimentation and optical rotatory measure- 
ments. The data that were obtained can be in- 
terpreted as showing that the growth hormone 
molecule loses some of its tertiary structure 
during acidification without there being any 
change in the helical content of the hormone. If 
the acidification is carried out in the absence of 
salt the transition is reversible. In the presence 
of salts containing various anions, the transi- 
tion is not reversible and insoluble aggregated 
forms are produced. 

In urea, the same type of transition seen at 
acid pH can be observed at neutral pH values.. 
At least 8 different configurations of growth, 
hormone were observed in all and the condi- 
tions under which these different forms can be 
transformed one into the other have been es- 
tablished. (Drs. H. Burger, P. G. Condliffe, and 
H. Edelhoch) 

Human Growth Hormone. Human growth 
hrmone was prepared from acetone pried pitui- 
taries by percolation. By using an immunoas- 
say procedure to follow the distribution of 
growth activity during gel filtration, two dis- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



191 



tinct forms of HGH having different molecular 
weights were demonstrated. Human TSH 
was obtained during percolation but in order 
to separate contaminating HGH an addi- 
tional step is required in the isolation process 
such as rechromatography on CM-C. (Drs. H. 
Burger, P. G. Condliff e and R. W. Bates) 

Pituitary Hormones in Human Plasma. 
The concentration of prolactin in blood in 
women 2 days post partum was 14 ± 3 
mU/ml (range 6 to 29) whereas that in normal 
controls was less than 5 mU/ml. There was no 
difference between the two groups in the con- 
centration of growth hormone in blood. This 
indicates a dichotomy between human prolactin 
and growth hormone. (Drs. R. W. Bates and J. 
Roth) 

Transplantable Pituitary Tumors. It 
was previously reported that injection of com- 
mon adrenal steroids into adrenalectomized 
rats with MtT tumors did not adequately re- 
produce the action of the adrenal gland in rats 
with MtT tumors upon organ size. In vitro stud- 
ies with enlarged adrenals from tumor bear- 
ing rats have shown that these adrenals pro- 
duce two unusual steroids. The same steroids 
have also been isolated from 1 liter of plasma 
of rats bearing the MtT. An attempt is now be- 
ing made to identify the steroids ; it is believed 
that they are 18-OH steroids. (Drs. R. W. 
Bates and D. Johnson) 

Metabolism of Adipose Tissue 

Glucose Metabolism in Isolated Fat 
Cells. Phospholipase C, the toxin of CI, 
perfringens, is an enzyme that catalyses the 
liydrolysis of phospholipids (primarily lecithin 
and sphingomyelin) to diglycerides (or sphin- 
gosine) and phosphorylcholine. This enzyme 
added at low concentrations caused an eleva- 
tion of glucose uptake by isolated fat cells to 
the same level observed when fat cells are incu- 
bated with insulin. Fatty acid synthesis and 
other parameters of glucose metabolism which 
are stimulated by insulin were similarly in- 
creased in fat cells treated with phospholipase 
C. Incorporation of amino acids into protein, 
thought by some investigators to be a specific 
^action of insulin, was also stimulated by the en- 
:zyme to the same degree observed with insulin. 



Pyruvate oxidation and incorporation into pro- 
tein of fat cells were stimulated by both insulin 
and phospholipase C. 

The level of hexokinase in the cell, the first 
enzyme involved in the metabolism of glucose, 
was unaffected by insulin or phospholipase C 
treatment and was found not to be the rate lim- 
iting step in glucose utilization by the fat 
cell. It was also found that complete inhibition 
by oligomycin of ATP synthesis in fat cell mi- 
tochondria did not affect the ability of insulin 
or phospholipase C to increase glucose utiliza- 
tion by the fat cell. These results suggested 
that insulin and phospholipase C did not affect 
the mitochondria, the principal source of en- 
ergy in this cell. 

It was possible to demonstrate, by the use of 
glucose analogues, that both insulin and phos- 
pholipase C increased the rate of glucose trans- 
port across the fat cell plasma membrane. 
These studies also revealed that the transport 
of glucose by the fat cell is a carrier-mediated 
process, i.e., transport is not by simple diffu- 
sion but is mediated by some process that is 
stereospecific and which has a selective affinity 
for different sugars. Phospholipase C neither 
altered the carrier properties of the transport 
system nor the ability of insulin to stimulate 
the transport system. Insulin and phospholi- 
pase C did not alter the affinity of the carrier 
for glucose but only the rate of penetration of 
the sugar. 

It was also found that increasing the concen- 
tration of phospholipase C in the incubation 
medium caused an apparent loss in the re- 
sponse of the fat cell to insulin. This apparent 
loss in response was actually the result of cell 
destruction as manifested by the release of sol- 
uble glycolytic enzymes to the medium and by 
the appearance of fat droplets that were re- 
leased from the cell. Addition of ATP and TPN 
to the partially or totally disrupted cells com- 
pletely restored glucose oxidation via the pen- 
tose pathway to the levels observed when intact 
cells were treated with insulin. This is further 
evidence that hexokinase is not rate limiting 
for glucose utilization. More importantly, these 
findings suggest that manifestation of insulin 
action on glucose utilization requires the pres- 
ence of an intact cell. 



192 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Lysis of the fat cell by phospholipase C ac- 
tion was not unexpected since a number of stud- 
ies have shown that this enzyme causes the 
lysis of red cells due to its action on the phos- 
pholipids of the red cell membrane. Indeed, this 
can be taken as evidence that the fat cell con- 
tains a similarly constituted plasma membrane. 
(Drs. M. Rodbell and G. de Cingolani) 

Effect of Growth Hormone and Gluco- 
corticoids ON ISLATED FAT CELLS. Severe 
ketosis, hyperlipemia and fatty livers develop in 
well-nourished rats when they are 'totally' pan- 
createctomized. Earlier studies in this labora- 
tory showed that the above did not develop if 
the rats were depleted of body fat or if they 
were deprived of the pituitary or adrenal 
glands. Subsequent studies showed that the 
glucocorticoid hormone was the only one needed 
for the development of ketosis in the hypophy- 
sectomized-pancreatectomized rat. However, if 
the rats had small amounts of insulin remaining 
in the tissues growth hormone was also re- 
quired. It was suggested that glucocorticoids 
and growth hormone produced the above effects 
by stimulating mobilization of lipid from adi- 
pose tissue to the liver. 

The effect of growth hormone and dexameth- 
asone on fatty acid release was studied in iso- 
lated fat cells prepared from adipose tissue of 
fasted normal rats. The hormones had very 
little effect when they were added separately. 
When the hormones were added together, how- 
ever, they markedly stimulated the release of 
both fatty acids and glycerol. The concentra- 
tion of hormones needed for these effects was 
0.016 ;u.g/ml for dexamethasone and 0.001 to 
0.01 ixg/ml for growth hormone; a maximal 
effect was obtained when the growth hormone 
concentration was 0.1 /^g/ml. 

It was also found that addition of very small 
amounts of insulin, 1 to 10 /lU/ml, blocked 
completely the lipolj^ic action of grovsrth hor- 
mone and dexamethasone. The antilipolytic ac- 
tion of insulin did not require glucose. 

The lipolytic action of growth hormone and 
dexamethasone was slow in onset ; no effect was 
see until after 1 hour of incubation. This de- 
layed effect was in marked contract to the rap- 
id effect of the other known lipolytic hor- 
mones: epinephrine, ACTH, TSH and gluca- 
gon. The slow onset suggested that synthesis of 



protein or RNA might be required for the lipo- 
lytic action of growth hormone and dexametha- 
sone. It was found that the lipolytic action of 
growth hormone and dexamethasone was 
blocked by puromycin (10-*M) and actinomy- 
cin-D (lO-'M) but not by the aminonucleoside 
of puromycin (10-^M). Puromycin (10-*M) 
also blocked the incorporation of labeled histi- 
dine, leucine and glycine into protein and actin- 
omycin-D (10"'M) blocked incorporation of la- 
beled uridine into RNA. Actinomycin-D also 
blocked the small stimulatory effect of growth 
hormone and dexamethasone on incorporation 
of labeled histidine and leucine into protein. 
When actinomycin-D was added two hours 
after growth hormone and dexamethasone, it 
did not alter the lipolytic action of these hor- 
mones during the subsequent two hours. If the 
only effect of actinomycin-D, added to 10"'M, 
is to inhibit RNA synthesis, the above observa- 
tions indicate that growth hormone and dexa- 
methasone accelerate lipolysis in the fat cell by 
stimulating RNA synthesis. 

The above findings clearly demonstrate that 
growth hormone and glucocorticoids added to- 
gether stimulate fatty acid release from fat 
cells and that this response can be blocked by 
insulin. In view of the very low hormone con- 
centrations needed for the in vitro effects, it is 
likely that these studies reflect the primary 
mechanism for mobilizing fatty acids in the 
living animal; the process is stimulated by an 
interaction of growth hormone and glucocorti- 
coids and is inhibited by insulin. (Drs. J. N. 
Fain, V. Kovacev and R. 0. Scow) 

Effect of Hormones on Fatty Acid Re- 
lease IN vivo. A technique has been developed 
in this laboratory for studying the effect of hor- 
mones on fatty acid release in viro in the rat. 
The method consists of collecting blood from 
the left parametrial fat body via the uterine 
vein and blood from the saphenous artery. The 
rate of fatty acid release is determined as the 
venous-arterial difference in plasma FFA con- 
centration and the rate of blood flow from the 
fat body. Fatty acid release (uEq/(g fat body 
X min) ) averaged 0.005 in fed rats, 0.03 in 
rats fasted 1 day, 0.06 in rats fasted 2 days, 
and 0.09 in rats 'totally' pancreatectomized 
for 1 day. Insulin, 2 units subcutaneously or in- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



193 



travenously, lowered blood glucose, blood 
ketone bodies and FFA release in pancreatecto- 
mized rats. In normal rats faster 2 days, how- 
ever, insulin injected, with or without glu- 
cose, lowered blood glucose and ketone bodies 
but had no effect of FFA release. Infusion of 
glucagon at 5.0 /xg/min, but not at 2.5 g/min, 
stimulated FFA release in insulin-treated pan- 
createctomized rats. The results suggest that 
insulin can stimulate the alpha cells of the pan- 
creas to secrete glucagon and that this effect is 
not mediated through the blood glucose level. 

The effect of growth hormone and dexameth- 
asone on fatty acid release in vivo was also 
studied with this technique. Injection of these 
hormones increased FFA release within 40 min- 
utes in rats fasted for 1 day. There was no 
significant increase in blood ketone body con- 
centration until 3 hours after injection. The 
hormones did not affect the blood glucose con- 
centration. (Drs. V. Kovacev and R. 0. Snow) 

Experimental Diabetes 

Pituitary Diabetes in Rats. A trans- 
plantable tumor that produces elevated blood 
levels of growth hormone, ACTH and prolactin 
induces diabetes in the host rat if 40% or more 
of the pancreas is removed. Histological exam- 
ination of the pancreases of these rats by Dr. 
Lacy of Washington University has shown that 
p cells are degranulated and vacuolated in 
those rats which are diabetic. Based on these 
findings studies have been started in rats with 
8.0 % of the pancreas removed to determine the 
role of growth hormone, ACTH and adrenal 
steroids in the induction of diabetes. The dose- 
response curves obtained so far indicate that 
growth hormone and ACTH are of equal im- 
portance and act synergistically. The current 
concept is that the adrenal hormones play only 
a permissive role. (Drs. R. W. Bates and R. 0. 
Scow) 

Ketosis. Some of the factors affecting 
ketone body formation in the liver were studied 
with the collaboration of Dr. Otto Wieland in 
Munich, Germany. Studies with liver slices 
confirmed the recent report that addition of 
calcium ion augmented the formation of ketone 
bodies from endogenous substrate. Oxidation 
of exogenous substrate (octanoate), however, 



was not increased by the addition of calcium to 
the medium. It may be that the hepatic lipases 
which hydrolize endogenous triglycerides and 
phospholipids are activated by calcium. This 
will be investigated further in the near future 
and may assist in understanding the mecha- 
nism of turnover of the complex lipids in the 
liver slice. 

It was found that addition of fructose to 
liver slices decreased the formation of ketone 
bodies from octanoate by diverting the octa- 
noate carbon to fatty acid and CO^ formation. 
Conversely the addition of octanoate to liver 
slices metabolizing fructose diverted fructose 
carbon from fatty acid and CO2 formation to 
ketone bodies without decreasing the amount 
of fructose utilized. The quantitative interrela- 
tion between fatty acid oxidation and the me- 
tabolism of carbohydrate in the liver awaits 
kinetic studies on the common intermediates of 
both systems. 

In a collaborative study with Dr. Philip Felts 
in Munich it was found that increased fatty 
acid exidationdue to feeding of fat or diabetes 
mellitus was not associated with decreased lev- 
els of citrate in the liver as had been found by 
others. The level of acyl-Co-A in the liver was 
directly related to the fat content of the liver 
and the ketonemia. High levels of acyl CoA 
were not accompanied by decreased citrate le- 
vels as would be expected from the in vitro ob- 
servation of Weiss and Wieland. They found 
that acyl-CoA is a potent inhibitor of the con- 
densation of acetyl-CoA and oxalacetate to 
form citrate. Thus, the suggested regulatory 
role of acyl-CoA on hepatic metabolism was not 
confirmed in vivo. (Dr. S. S. Chernick) 

Hepatic Fatty Acid Synthesis. It is well 
known that hepatic synthesis of long chain 
fatty acids (LCFA) is dependent on insulin. 
Many workers have concluded that the im- 
paired synthesis observed in fasting animals is 
also the result of insulin lack, secondary to de- 
creased secretion of the hormone. A study was 
made recently of the effect of 'total' pancrea- 
tectomy on fatty acid synthesis in the liver of 
fasting rats. Synthesis was measured by the 
amount of labeled acetate incorporated into 
fatty acids in liver slices and in soluble enzyme 
preparations of liver. The latter, prepared by 
homogenization and centrifugation at 100,000 



194 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



X g was fortified with ATP, MgCU, creatine 
phosphate and citrate. 

It was found that synthesis of LCFA was 
markedly impaired in liver slices and soluble 
enzyme preparations from rats fasted for 40 
hrs. Synthesis was also reduced in slices from 
fasted pancreatectomized rats whereas synthe- 
sis in soluble enzyme preparations of livers 
from diabetic rats was only slightly less than 
that from normal fed rats. Addition of glucose 
6-PO4 accelerated LCFA synthesis in enzyme 
preparations from all three groups of rats. 
Maximal synthesis in the presence of glucose 
6-PO4, however, was 8-4 times higher in 
preparations from fasted diabetic than from 
fasted normal rats. Administration of dexa- 
methasone in vivo increased LCFA synthesis in 
soluble enzyme preparations of liver, but not in 
liver slices, of fasted normal rats. At the pres- 
ent time it is not known what factors caused 
the difference on LCFA synthesis in the soluble 
enzyme preparations; between fasted normal 
and fasted pancreatectomized rats. It is conceiv- 
able that the difference may be due in part to 
increased secretion of glucocorticoids in the 
operated rats since administration of dexameth- 
asone significantly increased LCFA synthe- 
sis in the soluble enzyme preparation but not 
in liver slices. (Drs. R. 0. Scow and E. Urgoiti, 
with Dr. R. 0. Brady, NINDB) 

LABORATORY OF CHEMISTRY 
Section on Carbohydrates 

Synthesis of Nucleosides. The majority of 
naturally-occurring nucleosides are typified by 
the ribonucleosides in which the aglycons are 
attached trans to the C-2' hydroxyl group of 
the sugar moieties; for convenience, one may 
call them "trans nucleosides." The synthesis of 
substances of this class is readily effected 
through the tri-0-acylglycofuranosyl halides 
owing to the fact that the acyl group at C-2 of 
the sugar derivative participates in the dis- 
placement of the halogen atom, ensuring a 
trans attachment of the aglycon. A few cis nu- 
cleosides have been encountered in nature. Of 
these, one may cite 5,6-dimethyl-l-a-D-ribo- 
furanosylbenzimidazole (of-ribazole) , a moiety 
of the vitamin B12 molecule, and 9-jS-D-arabino- 
furanosyladenine (spongoadenosine) which oc- 



curs in the anaerobic sponge Cryptotethya 
crypta. The remarkable cytotoxicity of this 
latter substance has attracted considerable at- 
tention and aroused interest in other ;S-D-ara- 
binofuranosyl nucleosides. However, a practi- 
cal pathway for the chemical synthesis of sub- 
stances of this class was lacking until Dr. C. P. 
J. Glaudemans discovered that the readily 
preparable halide, 2,3,5,-tri-O-benzyl-D-arabino- 
furanosyl chloride, condenses with N-benzoy- 
ladenine to give (after removal of masking 
groups) a substantial yield of spongoadenosine. 
More recently. Dr. J. D. Stevens showed that 
tri-0-benzyl-D-ribofuranosyl chloride may sim- 
ilarly be used for the synthesis of a-ribazole. 
These new syntheses, involving the use of non- 
participating groups in glycosyl halides, ap- 
pear to offer much promise for the sjoithesis of 
cis nucleosides and a variety of other difficulty 
accessible carbohydrate derivatives of biochem- 
ical and medical importance. However, in 
order to make the most effective use of this 
synthetic approach, we need to know the mech- 
anism whereby the halogen in these gylco- 
furanosyl halides is displaced as well as the 
stereochemical features of the reaction. Unfor- 
tunately the two halides mentioned are amor- 
phous and thus not ideally suited for me- 
chanistic studies. Dr. Glaudemans has, how- 
evei', succeeded in synthesizing the two anom- 
eric 2-0-nitro-3,5-di-0-p-nitrobenzoyl-D-arabi- 
nofuranosyl chlorides; these are crystalline sub- 
stances and represent the first known pair of 
anomeric glycosyl halides which lack a partici- 
pating group at C-2. Studies of the solvolysis 
of these halides show that they both give pre- 
dominantly cis products, a finding which sug- 
gests that cis nucleosides may generally be 
preparable from glycofuranosyl halides lacking 
nonparticipating groups regardless of the 
anomeric configuration of such halides. 

Methods for the chemical synthesis of deoxy- 
ribonucleosides still leave much to be desired. 
By way of exploring novel approaches to 
this problem. Dr. R. K. Ness synthesized 
3,5-di-0-benzoyl-l,2-dideoxy-D-er2/i/iro-pento- 
f uranose-1-ene, the first f uranose-related glycal. 
Unfortunately, the tendency of this structure 
to degrade to furan derivatives has thus far 
prevented its use for the synthesis of deoxy- 
nucleosides. Theoretical considerations sug- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



195 



gested that the 3,5-di-0-2)-anisoyl analog would 
prove more stable; Drs. R. K. Ness and M. 
Haga have succeeded in synthesizing this 
analog and have found that it is indeed more 
stable than the di-0-benzoyl derivative. 

Synthesis of Substances Related to 
Certain Mucoproteins. It has been postu- 
lated that the alkali-labile N-acetyl-D-galactos- 
amine residues in ovine submaxillary gland 
mucoprotein are attached as esters between 
C-1 of the aminosugar and the nonpeptide- 
bonded carboxyl groups of aspartic and glu- 
tamic acids. For this reason it is of interest to 
examine the properties of the C-1 esters of the 
N-acetyl hexosamines. Dr. R. Harrison has de- 
veloped a synthesis whereby he has been able 
to prepare various C-1 esters of N-acetyl-D- 
glucosamine and N-acetyl-D-galactosamine. Dr. 
T. D. Inch has studied some of the properties 
of these substances and found the equatorial 
esters to be markedly more labile than the axial 
esters. 

Studies on the Synthesis of Starfish 
Sialic Acid. Dr. L. Warren, while in the Lab- 
oratory of Biochemical Pharmacology of 
NIAMD, discovered a new form of sialic acid 
in the starfish Asterias Forbesi and postulated 
that it has the structure of N-glycolyl-8-0- 
methylneuraminic acid. In preliminary studies 
of possible pathways for the synthesis of this 
substance, two approaches have been pursued. 
The first approach involved the synthesis of 4- 
0-methyl-D-arabinose, a six-carbon fragment 
representing carbon atoms 5-9 of the sialic 
acid. Mr. H. W. Diehl succeeded in synthesizing 
4-0-methyl-D-arabinose from methyl a-D-ara- 
binofuranoside via 2,3,5-tri-O-benzyl-D-arabi- 
nofuranose. The synthesis itself is of some 
general interest as representing a method 
whereby substituents may be inserted on the 
carbon atom which normally denotes the 
oxygen atom of the ring structure of a sugar. 

The second approach to the synthesis of 
starfish sialic acid has involved studies of the 
selective substitution of reactive groups in N- 
acetyl-D-mannosamine. Direct benzylation of 
N-acetyl-D-glucosamine and of N-acetyl-D-ga- 
lactosamine was shown by Dr. R. Harrison to 
yield the corresponding benzyl 2-acetamido- 
3,4,6-tri-0-benzyl-2-deoxy-hexopyranosides. Dr. 
-J. R. Plimmer has now found that applica- 



tion of the same process to N-acetyl-D-manno- 
samine causes epimerization, the product being 
benzyl 2-acetamido-3,4,6-tri-0-benzyl-2-deoxy- 
D-glucopyranoside. This difficulty has now 
been overcome and the synthetic pathway is be- 
ing pursued further. 

Higher-Carbon Sugars. The studies of the 
higher-carbon sugars and other carbohydrates 
in the roots of Primula officiruilis Jacq. were 
continued by Dr. R. Begbie. In addition to pri- 
meverose (6-0-j8-D-xylosyl-D-glucose) , and 
volemitol (D-g^Zi/cero-D-mcmno-heptitol) which 
had been reported many years ago, and sedo- 
heptulose (D-aZiro-heptulose), D-man%o-heptu- 
lose, and /3-sedoheptitol (D-glycero-D-gluco- 
heptitol) which had been mentioned in the 
preceding report, the following substances 
were isolated and identified; glycerol, erythri- 
tol, D-xylose, xylitol, mi/o-inositol, D-glycero- 
/J-D-grZ^co-heptose (crystalline for the first 
time) , D-glycero-D-manno-heTptose, D-aZZo-hep- 
tulose, D-aZfro-3-heptulose, and the same octu- 
loses and nonuloses that had been isolated pre- 
viously from the avocado, namely, D-glycero- 
D - tnanno - octulose, D -glycero - L - galacto - octu- 
lose, D-erythro-L-gluco-noYiulose, and B-ery- 
thro-h-galacto-nonulose. Studies on the carbo- 
hydrate constituents of Sedum species and 
of the avocado by Dr. H. H. Sephton, and 
on Pichi tops (the foliage of Fabiana imbri- 
cata) with the assistance of Mr. Tracy were 
continued. A similar study of the roots of Se- 
dum spectabile was begun by Dr. H. J. F. 
Angus. 

The phenyl a- and /3-sedoheptulosides men- 
tioned in the preceding report have been found 
by Mr. E. Zissis to be hydrolyzed very readily 
by acids, even at room temperature. They are 
extremely sensitive toward alkalies : methanolic 
sodium methoxide converts them into methyl a- 
sedoheptuloside (which was synthesized inde- 
pendently for comparison) and dilute aqueous 
potassium hydroxide converts them very rap- 
idly at room temperature into sedoheptulose 
and phenol. 

Miscellaneous Researches. Through a 
transvinylation reaction, 2,3,4,6-tetra-O-ben- 
zyl-D-glucopyranose has been converted to vi- 
nyl 2,3,4,6-tetra-O-benzyl-D-glucopyranoside by 
Dr. C. P. J. Glaudemans. The hydrogenation of 



196 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



this substance affords ethyl D-glucopyranoside 
(predominantly the a anomer) . 

l,5-Anhydro-3,4-di-0-benzoyl-2-0-thioben- 
zoyl-D-arabinitol, the first thionobenzoate of a 
carbohydrate derivative, has been synthesized. 

Nucleosides containing D-xylofuranose moie- 
ties are increasingly being used as interme- 
diates for the preparation of a wide variety of 
synthetic nucleosides. Mr. H. W. Diehl has 
markedly improved the preparation of the an- 
omeric D-xylofuranose tetrabenzoates, com- 
pounds which are required for the synthesis of 
D-xylofuranosyl nucleosides. 

Dr. H. H. Sephton has demonstrated that va- 
por phase chromatography may be used for the 
separation and determination of the hydrolysis 
products of a methylated xylan as their tri- 
methylsilyl derivatives. This exploratory work 
constitutes an important improvement in the 
techniques for the elucidation of polysaccha- 
ride structure. 

Section on Medicinal Chemistry 

Evaluation of Analgesics. Dose-range- 
finding experiments were performed on 63 
drugs (many from universities and the phar- 
maceutical industry in the U. S. and abroad). 
There were 69 complete assays by the Eddy 
hot-plate method and probit analysis of the 
data and 50 acute toxicity experiments. During 
the course of the year's work, it was discovered 
that the so-called "cleaner" white mouse fur- 
nished by the Animal Production Unit since 
December 1962 is far more sensitive to CNS 
drugs than the old GP mice. Revised EDso val- 
ues for standard analgesics such as morphine, 
codeine and pethidine will be published. The 
unit continues to cooperate with and advise the 
pharmacology unit of the University of Michi- 
gan and the Addiction Research Center on ad- 
diction studies and serve in an advisory capac- 
ity for the Bureau of Narcotics, the Committee 
on Drug Addiction and Narcotics, FDA and 
WHO. (E. L. May, and N. B. Eddy) . 

1-AZONIABICYCLO [3,1,0] HEXANE, A NOVEL 

Ring System, and Precursor of a ^e-BENzo- 
MORPHAN. 2-Benzyl-3,4-dibromo-l,3,4-trimeth- 
ylpiperidine has been isolated as the hydrobro- 
mide salt (two isomers from the bromination of 



2-benzyl-l,3,4-trimethyl-l, 2, 5, 6-tetrahydropyri- 
dine. These were transformed into 6-benzyl-4- 
bromo- 1, 4, 5-trimethyl- 1-azoniabicyclo [3, 1, 0] 
hexane perchlorates (two isomers) (I) epimeric 
at the benzyl substituent. Both structures are 
based on a irans-addition of bromine to the 
double bond with subsequent backside displace- 
ment of the bromine on position 3 by nitrogen. 
Models indicated that in only one of these is the 
benzene ring in position to bond with the 4-posi- 
tion of the piperidine ring to give a cyclobenzo- 
morphan which could be expected to yield ^- 
2,5,9-trimethyl-6,7-benzomorphan. This conver- 
sion was realized for this isomer by cleavage of 
the aziridinium ring with lithium aluminum 
hydride. With potassium acetate the I isomers 
were converted to a 2- (hydroxyphenethyl) 
3-methylenepyrrolidine (ring contraction) and 
a 3-hydroxy-4-methylenepiperidine. (E. M. 
Fry) 

4-SUBSTITUTED-8-METHYL-l-OXA-4,8-DIAZA- 
SPIRO [4, 5] DECANES (SPIROPIPERIDINOOXAZOLI- 

DINES. Whereas aromatic aldehydes react with 
2-aminoethanol to give Schiff bases to the almost 
complete exclusion of isomeric oxazolidines, 
with l-methyl-4-piperidone the spirooxazolidine 
derivative predominates. The use of mono-N- 
alkylated-2-aminoethanols gives exclusively ox- 
azolidines (II), of course. With aromatic alde- 
hydes, the II are sensitive to even traces of acid 
but can be characterized as picrate salts in dry 
non-polar solvents. The oxazolidines from N- 
methyl-4-piperidone are relatively stable in 
dilute acid. Ethanolamine and N-methyl-N- 
butyl- and N-(2-hydroxethyl)-, ethanolamines 
have been used and give the title compounds. 
Hydrogenation of either the Schiif bases or the 
oxazolidine isomers with 1 molar equivalent of 
hydrogen (platinum oxide) gives the corre- 
sponding ^-amino alcohol. The latter and pre- 
cursor oxazolidines are of interest as carci- 
nolytic antimicrobial anti-inflammatory and 
diuretic agents. (J. Harrison Ager) . 

Aldolase Inhibitors. Treatment of dieth- 
yl 2,5-dioxoterephthalate with excess lithium 
aluminum hydride (LAH) gives trans-2,5-di- 
hydroxy-l,4-dimethylenecyclohexane (III) the 
structure and configuration of which were as- 
signed on the basis of facile absorption of two 
molar equivalents of hydrogen (platinum ox-- 



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197 



ide), n.m.r. data and analogy with the course 
of LAH reduction of other ^S-keto esters. Hy- 
droxylation of I to 1,2,4/5-tetrahydroxycyclo- 
hexanedimethanol-1,4 (IV) was achieved ulti- 
mately in a reproducible 52% yield with a 
combination of osmium tetroxide and hydrogen 
peroxide. In preliminary tests, IV showed activ- 
ity against P1798 lymphosarcoma. (James G. 
Murphy) . 

immunochemistry-polyethylenimine and 
Synthesis of 4-p-Methoxyphenyl-1-vinyl- 
PYRROLIDONE. Viscosity studies on polyethyl- 
enimine (PEI) polymers indicate that the dis- 
coloration noted for fractionated material is due 
to an alteration of chain structure. PEI polymer 
binds copper to the extent of one Cu to 5 nitro- 
gens. The end-point of the binding and a quan- . 
titative assay for PEI polymer are determined 
by a change in the UV spectrum. This copper 
chelation has apparently caused only a small 
change in viscosity which probably means that 
the polymer is highly branched. In a strictly 
linear polymer, a strong coordinating agent will 
contract the chain and produce a marked 
change in viscosity. 

The synthesis of 4-2?-methoxyphenyl-l-vinyl- 
pyrrolidone was achieved in 8 steps consisting 
of (1) Reformatsky cyclization of w,4-diacetoxy- 
acetophenone in the presence of ethyl bromo- 
acetate; (2) reduction with 5% Pd-C; (3) dia- 
zomethane methylation; (4) reaction with 
N,N-dimethylethylene diamine; (5) treatment 
with thionyl chloride; (6) treatment with 
K-f-butoxide ; (7) conversion to the methiodide ; 
(8) Hofmann elimination. Yields are 50% and 
above in all steps. This monomer will be homo- 
and co-polymerized after or before 0-demethyl- 
ation. The vinji group seems sluggish toward 
catalytic reduction. (T. D. Perrine). 

Instrumentation. In addition to complet- 
ing a set of 6 high-temperature bank gas chro- 
matographs, in cooperation with the Kontes 
Glass Company, an addition funnel has been 
devised that will meter the flow of a liquid 
reactant into a reaction mixture either at a 
constant rate or at a constant reaction temper- 
ature. (T. D. Perrine) . 

Synthesis of Bis-4- (cis-2,5-DiMETHYL-l-FOR- 
MYL) Piperazinylmethane. Careful reaction 



of cis-2-5-dimethylpiperazine with ethyl for- 
mate gives l-formyl-cis-2,5-dimethylpiperazine 
which, on reaction with aqueous formaldehyde 
in boiling benzene affords the title compound, 
apparently effective in controlling certain 
types of mammary cancer. (E. L. May) . 

6,7-Benzomorphan Research. Optical res- 
olution of a:-5,9,-diethyl-2'-hydroxy-2-methyl-6, 
7-benzomorphan (V), morphine-like for pain 
relief in animals and man with lower toxicity 
and much lower (almost no) drug dependence 
capacity in monkeys than morphine, gives a 
Ze-yo-isomer which is twice as potent as and 
much less toxic than the racemate. The 
dextr o-isomer, although much less effective 
than its antipode is, nevertheless, codeine-like 
in analgesic activity. Surprisingly too, its phys- 
ical dependence capacity in the monkey is in- 
termediate, though nil for V. This indicates an- 
tagonism between the antipodes. The levo- 
isomer is being studied. (E. L. May). 

a - 5,9-Dimethyl- 2'-hydroxy-2-trideuteriometh- 
yl-6,7-benzomorphan (VI) has been synthesized 
by lithium aluminum deuteride reduction of 
a-2, 2'dicarbethoxy-5, 9- dimethyl - 6,7 -benzomor- 
phan in turn prepared from the corresponding 
nor phenolic compound with ethyl chloroform- 
ate. The analgesic activity of VI is about % 
that of the N-methyl parent while the pKa is 
8.53 compared to 8.38 for the N-CH3. (T. 
Ishimaru and E. L. May) . 

Aluminum bromide cyclization of 3,4-dieth- 
yl-2-{p- hydroxybenzy 1 ) -2 -methyl- 1 , 2 , 5 , 6-tetra- 
hydropyridine hydrochloride (VIII) gives, in 
addition to about equal amounts of a- and jS- 
benzomorphan, l,5-dimethyl-4-ethyl-7-hydroxy- 
l,2,3,4,5,10,ll,12-octahydrobenzo(g)quinolineas 
the predominant product (27% yield). Its 
constitution was determined by n.m.r. mass 
spectra, by mass, UV, and n.m.r. spectra of its 
Hofmann elimination product (VIII) and by 
isolation of 7-methoxy-l-methylnaphthalene 
after aromatization of VIII. This predominant 
product, resulting from a shift of the 
3,4-double bond to the 3-exo position followed 
by cyclization is nearly as potent as morphine 
despite its structural nonconformities. (E. L. 
May, B. C. Joshi, A. E. Jacobson with H. M. 
Fales and J. W. Daly) . 



198 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Similar cyclization of the 3-ethyl-4-methyl 
compound corresponding to VII gave, in addi- 
tion to the expected benzomorphans, a by-prod- 
uct identical to one obtained earher by 
aqueous HBr cyclization. On the basis of an 
n.m.r. spectrum this product appears to be not 
the azaanthracene structure but an indano 
compound resulting from 5-membered ring clo- 
sure of the tetrahydropyridine. (J. H. Ager) . 

Synthesis of 5-butyl-, amyl-, and hexyl-2'-hy- 
droxy-2-methyl-6,7-benzomorphan and a-5,9- 
dimethyl - 2 - hexyl-2'-hydroxy-6,7-benzomorphan 
completes each series from methyl to hexyl. In 
the 5-alkyl series, Ci, C5 and Ce confer potency 
comparable to morphine ; activity is nil for C2, 
€3 and C4. The N-propyl compound is one of the 
most potent morphine antagonists known. (B. 
C. Joshi, C. Chignell and E. L. May) . 

Replacement of 2'-0H of the benzomorphan 
nucleus with nitro, amino, chloro or fluoro has 
a detrimental effect on both analgesic potency 
and toxicity. During the course of synthesis of 
the 2'-chloro compound, two isomeric by-prod- 
ucts were uncovered in the Stevens rearrange- 
ment of l-39-chlorobenzyl-l,3,4-trimethyl-l,2,5,- 
6-tetrahydropyridinium chloride which on the 
basis of infrared, n.m.r. and mass spectral data 
appear to be 4-p-chlorobenzyl-l,3,4-trimethyl- 
1,2,5,6,-tetrahydropyridine and 1,3,3,-trimethyl- 
2-p-chlorobenzyl-4-methylenepiperidine. (A. E. 
Jacobson) . 

A Naecotine Derivative. Reaction of co- 
tarnine, a cleavage product of the abundant 
opium alkaloid, narcotine, with 3-phenylpro- 
pylmagnesium bromide has given a good yield 
of 1- (3-phenylpropyI) -2-methyl-6,7-methylene- 
dioxy-8-methoxy-l,2,3,4-tetrahydroisoquinoline, 
codeine-like in analgesic activity and active 
against a resistant strain of Staphylococcus au- 
reus. It also appears to have a diuretic effect in 
mice. (A. E. Jacobson). 

Quinine and Quinidine Polygalactu- 
RONIDES. Reaction of quinine and quinidine 
(separately) with polygalacturonic acid (mol. 
wt. 40,000-80,000) in aqueous methanol, filtra- 
tion, vacuum evaporation to dryness and ex- 
haustive extraction of the residue with boiling 
acetone gave in each instance, white, sparingly 
soluble amorphous powders. These salts were 



prepared for Dr. N. R. Shulman for immunolog- 
ic studies in clinical hematology. (L.J. Sar- 
gent) . 

Unnatural Nucleosides. Unsuccessful at- 
tempts to construct the quinazoline-dione nu- 
cleoside system include (1) acylation of the 
glycosyl amine prepared from o-cyanoaniline 
and glucose with ethyl chloroformate led to the 
corresponding tetra-0-carbethoxy urethane 
which did not cyclize to the expected nucleoside 
with sodium ethoxide; (2) ethoxide treatment 
(for cyclization) of the product obtained in 
reacting 2,3,4,6-tetra-O-benzyl glycosyl chlo- 
ride with sodio-o-cyanophenylurethane; (3) 
condensation of the sodio derivative of 
2,4-quinazoline dione with acetobromoglucose 
in dimethylformamide. (L. J. Sargent) 

Dihydrocodeinone Isomers. Separation of 
the isomers (N closed to the 5 or 7 position) 
obtained in the alkaline cyclization of 1,7-di- 
bromodihydrocodeinone dihydromethine is 
achieved more effectively before catalytic re- 
moval of the 1-bromo substituent. A Favorskii 
cyclopropane intermediate which can be at- 
tacked by nucleophilic nitrogen at either the 5 
or 7 position, is suggested to account for the 
unexpected cyclization to position 5. (L. J. Sar- 
gent and B. C. Joshi). 

Section on Metabolites 

Batrachotoxin, the Strongest Venom 
Known. Further progress has been made on 
the problem of the structure of batrachotoxin. 
Three congeners, named batrachotoxinin A, B 
and C, have been isolated by preparative thin 
layer chromatography. A potential pyrrole 
system has been detected in batrachotoxin by 
the strong color reaction with Ehrlich's re- 
agent and ioara-dimethylaminocinnamaldehyde. 
Much of the toxic effect appears to be due to a 
selective cardiotoxicity, to judge from current 
pharmacological investigations by Dr. Lou 
Harris at the Sterling-Winthrop Research In- 
stitute. NMR and mass spectrophotometric 
data have now supplied all the information 
possible. In order to elucidate the structure x- 
ray crystallography will now be employed. Dr. 
John W. Daly is currently looking into the cul- 
tivation of suitable crystals of the methiodide, 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



199 



as well as jjara-iodophenylhydrazone derived 
from batrachotoxin and batrachotoxinin A. 

Structure and Synthesis of the Gram- 
icidins. By countercurrent distribution and 
redistribution the commercially available pep- 
tide antibiotic gramicidin was resolved on a 
preparative scale. The group of lipophilic pep- 
tides contained gramicidin A, B and C, each of 
which consisted of a pair of congeners, i.e., va- 
line-gramicidin (80-95%) and an isoleucine- 
gramicidin (5-20%). The sum of aromatic 
amino acids was always 4, namely 4 Try in 
gramicidin A, 3 Try-M Phe in gramicidin B 
and 3 Try + 1 Try in gramicidin C. A more hy- 
drophilic, strongly antibiotic group of peptides, 
designated gramicidin D, contained 5-6 addi- 
tional amino acids. Complete amino acid anal- 
yses, including time-dependence studies of hy- 
drolyses, were carried out over the entire 
range of 999 transfers (E. Gross). 

Gramicidin A is a linear N-acylated pentade- 
capeptide ethanolamide. Its N-terminal L-va- 
line or L-isoleucine is blocked by a formyl 
group which is cleaved by mild methanolysis at 
room temperature and easily identified as for- 
mic acid by gas chromatography or as formal- 
dehyde after reduction by the chromotropic 
acid assay. Reformylation of desformylgrami- 
cidin A, via 0-formylgramicidin A, gave back 
gramicidin A. Desformylgramicidin A was 
subjected to ten successive Edman degrada- 
tions under conditions modified to accommo- 
date the insolubility of its residual peptide 
fragments in water. The phenylthiohydantoins, 
obtained by cyclization with trifluoroacetic 
acid, were assayed by gas chromatography up 
to step 10. In addition, the residual peptides up 
to step 10 were hydrolyzed and their amino 
acids determined quantitatively. Selective N- 
bromosuccinimide (NBS) cleavage of the 
bonds following the four tryptophans in grami- 
cidin A releases aminoethanol and the NBS ox- 
idation product of leucyl-tryptophan, but no 
dioxindolalaninespw-olactone fragment expect- 
ed from a Try-Try sequence. These findings 
combined with the older observations suggest 
the sequence: HCO-L-Val-Gly-L-Ala-D-Leu-L - 
Ala-D-Val-L - Val-D-Val-L - Try-D-Leu-L - 
Try-D-Leu-L - Try-D-Leu-L - Try-NH-CH^- 



CH2-OH for valine-gramicidin A and HCO-L- 
Ileu . . . for isoleiicine-gramicidin A. Thin layer 
chromatography, optical rotatory properties in 
various solvents, molecular weight studies by 
ultracentrifugation and osmometry, are expli- 
cable in terms of easy association of at least 
two molecules of gramicidin A whose unprece- 
dented alternating sequence of (mostly hydro- 
phobic) L- and D-amina acids permits a char- 
acteristic secondary structure dependent on 
the nature of the solvent (R. Sarges) . 

Coupling of the octapeptide derivative Z-L- 
Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D- 
Val-Ns with the heptapeptide ethanolamide L- 
( H ) Try - D-Leu -L- Try -D-Leu-L-Try - D - Leu - L- 
Try-NH-CHo-CHo-OH, or, more conveniently, of 
the pentapeptide Z-L-Val(or L-Ileu) -Gly-L-Ala- 
D-Leu-L-Ala-OH with the decapeptide enthanol- 
amide R-L-Val(or L-Ileu)-Gly-L-Ala-D-Leu-L- 
Ala-D-Val-L-Val-D-Val-L-Try-D-Leu-Try-D- 
Leu-L-Try-D-Leu-L-Try-NHCHoCH^HO, hav- 
ing all the properties of N-carbobenzyloxydes- 
formyl (=Z), desformyl- (R = H), and (R = 
CHO) valine- or isoleucine-gramicidin (R. 
Sarges) . 

The volatile acidic fragment in the hydroly- 
sate of gramicidin B and C was identified as 
formic acid which, after reduction to formalde- 
hyde, was assayed by the chromotropic acid 
test. The primary sequence of gramicidin B 
(C), which in the sample examined consisted 
of 89% of valine-gramicidin B (C) and 11% of 
isoleucine-gramicidin B (C), was determined 
by a 14-step Edman degradation on desfor- 
mylgramicidin B (C) (obtained from gramicid- 
in B (C) by mild methanolysis) as HCO-L-Val 
( L-Ileu ) -Gly-L-Ala-D-Leu-L-Ala-D- Val - L- Val- 
L-Try -D-Leu-L-Phe ( L- Try ) - D - Leu - L- Try- D- 
Leu-L-Try-NH-CH^CH^-OH (R. Sarges). 

Interaction of Mercuric Acetate with 
the Indole Chromophore in Tryptophan 
Derivatives and Proteins. Indole reacts 
with two moles of mercuric acetate to give 
2,3-diacetoxy-mercuri-indole. Reaction of in- 
doles with excess mercuric acetate in 50% ace- 
tic acid results in a bathochromic shift of the 
spectrum. The spectral changes are useful in 
the detection of the indole grouping in the 
free state and bound as tryptophan in proteins. 



200 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Mercurated indoles regenerate the parent in- 
dole on reaction with thiols or strong cation ex- 
changers. Mercurated indoles retain the sus- 
ceptibility of the present indoles to oxidation 
by NBS, whereas phenols, such as tyrosine, be- 
come resistant to oxidation in the presence of 
excess mercuric acetate. The use of mercuric 
acetate for the selective oxidation of indoles, in 
the presence of phenols, with NBS is indicated. 
Electron-withdrawing groups on both the 2- 
and 3-positions of indole as well as halide ions 
prevent the normal spectral shifts seen with 
mercuric acetate (L. K. Ramachandran) . 

2-Acetamido- and 2-benzamido-allyglycine 
and methallylglycine peptides and amides are 
cleaved by heating at 80° in ethanol in the pres- 
ence of excess mercuric acetate. The amine 
component is released in yields up to 30%. The 
treatment of rufomycin A in similar fashion 
releases certain amino groups from linkage. 
The released amino groups belong to 2-amino- 
hexenoic acid, leucine, alanine and O-NO2 tyro- 
sine. At pH 5.5 and 80° Ce^% La++% Hg++, 
CO+% and Ca++ do not cause any rupture of 
the peptide bonds in rufomycin A. However, 
some of these ions catalyze the hydrolysis of 
about 40% of one bond at lower pHs. In the 
presence of HBr, HCl, and to a lesser extent 
HF, at room temperature the peptide bond 
formed by the amino group of 2-amino-hexen- 
oic acid, in 0-methyl-dihydrorufomycin A, was 
particularly labile (H. Iwasaki) . 

Reactivity of Tryptophan in Native 
AND Inactivated Enzymes. The extent of 
oxidation of bound tryptophan, in addition to 
being dependent upon pH (increasing as the 
pH is decreased), was also found to be very 
sensitive to buffer concentration and tempera- 
ture. 8-Chymotrypsin, inactivated by reaction 
with diisopropylphosphoryl fluoride (DFP), 
?)-nitrophenyl acetate or N-tosyl-L-phenylala- 
nine chloromethyl ketone were found to be sig- 
nificantly less reactive toward oxidation by 
NBS at pH's 5.5, 6.0 and 7.0 than chymotryp- 
sin itself. The zymogen was also slightly more 
resistant to oxidation at these pH's. Trypsin 
inactivated by reaction with DFP or with the 
naturally-occurring polypeptide inhibitor from 
bovine pancreas was also more resistant to 
NBS oxidation than native trypsin; the effect 
of inhibition on tryptophan-oxidizability being 



dramatic in the latter case. Trypsin, partially 
inhibited with synthetic polypeptides of L-glu- 
tamic acid and L-leucine or L-tyrosine did not 
exhibit any difference in the reactivity of its 
tryptophan from native trypsin. Thus the modes 
of inhibition of the natural and synthetic inhib- 
itors probably differ. a-Chymotrypsin in the 
presence of large excesses of the inhibitors, 
benzoic acid or N-acetyl-DL-phenylalanine, 
showed no detectable difference in tryptophan 
oxidizability when compared with a-chymo- 
trypsin in the absence of the inhibitors (T. F. 
Spande and N. M. Green) . 

A Water-soluble Protecting Group for 
Peptide Synthesis. For the synthesis of 
acid-labile peptides, especially for those that 
contain tryptophan, the water-soluble acetyl- 
methionyl group provides good protection and is 
easily removed by cyanogen bromide (E. 
Gross) . 

Selective Cleavage of Proteins by 
Cyanogen Bromide. Cyanogen bromide has 
been used for the selective cleavage of the fol- 
lowing proteins: brazil nut allergen, chymo- 
trypsin, carbonic anhydrase, glutamic dehydro- 
genase, pepsin, trypsin and rhodopsin. Current 
investigations aim at the cleavage of certain 
fractions from human globulin, especially from 
a^-globulin (E. Gross, and R. Axen) . 

Cyanogen Bromide Cleavage op S- 
Methylcysteine Peptides. Cysteine: A/'-acyl 
derivatives of the lower homologue of methio- 
nine, i.e., S-methylcysteine, react also with cyan- 
ogen bromide. The peptide bond in N-acylamino- 
acyl-S-methylcysteine is subject to cleavage 
by cyanogen bromide. The reaction proceeds by 
participation of the peptide carbonyl of the 
amino acid that precedes S-methylcysteine. An 
oxazolidinium salt is formed as intermediate 
and easily hydrolyzed to O-(N-acylaminoacyl)- 
serine, the commonly encountered product of 
the N-»0-acyl shift of N-acylserine. Special 
reaction conditions are required for the hy- 
drolysis of the 0-acyl derivative. Model peptides 
such as N-acetylglycyl-, N-acetylvalyl-, N-acetyl- 
prolyl-, and N-acetylleucyl-S-methylcysteine 
were cleaved in yields of over 60%. The re- 
action with cyanogen bromide is not restricted 
to the S-methyl derivative of cysteine. Numer- 
ous S-alkyl-N-acyl derivatives of cysteine were 



NATIONAL INSTITUTE OP ARTHRITIS AND METABOLIC DISEASES 



201 



successfully reacted with cyanogen bromide. 
The general applicability of the cyanogen bro- 
mide reaction to S-alkyl-N-acylcysteine offers 
convenient access to many 0-acyl derivatives of 
serine (E. Gross). 

Synthesis and Biosynthesis of De- 
hydrobufotenine and role of the enzymes 
Involved. The synthesis of the tricyclic sero- 
tonin metabolite in toads, namely, dehydro- 
bufotenine, has been achieved via 5-ben- 
zyloxy-6-nitrogramine and five subsequent 
steps. The conversion of tritiated precursors 
such as tryptophan, 5-hydroxytryptophan, N- 
methylserotonin and bufotenine in the toad 
(Bufo marinus) has been investigated. The fol- 
lowing enzymes play a significant part in this 
conversion: aromatic decarboxylase, trypto- 
phan-5-hydroxylase, and a special enzyme 
system that catalyzes dehydrogenation to the 
trycyclic dehydrobufotenine. Attempts to char- 
acterize and isolate these enzymes are in prog- 
ress (S. Senoh, J. Daly, and C. Creveling). 

Manipulation of the Biosynthesis of 
Collagen through Fluoroprolines. cis- 
and ira.?is-4-Fluoro-L-prolines were prepared 
stereospecifically by Sn2 displacements of tosyl- 
oxy groups of natural and allo-N-carbobenzyl- 
oxy-0-tosylhydroxy-L-proline methyl esters 
with potassium fluoride. The pure fluoropro- 
lines were catalytically tritiated to uniformly 
labeled cis- and ira7!,s-4-fluoro-L-prolines. Both 
fluoroprolines were incorporated into chick em- 
bryo collagen and apparently more so than into 
normal protein. Only iraws-fluoroproline bound 
in procollagen was hydroxylated by proline- 
4-hydroxylase to tritiated hydroxyproline, 
which was isolated. The fluoroprolines make 
possible the study of collagen biosynthesis in 
chick embryos and raw paw pouches. The ma- 
nipulation of proline hydroxylase and the com- 
petitive incorporation of fluoroproline into 
procollagen will be studied in its effect on tissue 
regeneration and wound healing (Y. Fujita). 

New Amino Acids and Pharmacodynamic 
Amines. A microsomal enzyme system has 
been studied in considerable detail which is ca- 
pable of converting a wide variety of phenols 
to catechols. Among the transformations of 
special interest are the conversion of N-acetyl- 
serotonin to N-acetyl-5,6-dihydroxytryptamine 



and the formation of catecholamines from 
phenolic precursors, such as dopamine from 
tyramine and (nor) -epinephrine from (nor) 
synephrine. Metanephrine is hydroxylated to a 
trihydroxy-compound related in structure to 
mescaline. This establishes a metabolic inter- 
action of (nor) epinephrine and mescaline. In- 
hibitor studies have indicated that more than one 
enzyme is involved in these hydroxylations. The 
hydroxylation has been shown to occur with re- 
placement of halogen in certain compounds. 
Some correlations of structure versus activity 
bearing on the mechanism have been made. 

2,3-Dihydrotryptophan has been prepared by 
two different reductions, one involving the bis- 
trifluoroacetyl derivative. Tritiated material 
was demonstrated to be transported into brain, 
to form 2,3-dihydroindoleacetic acid probably 
via decarboxylation of the intermediate dihy- 
droindolepyruvic acid. This amino acid was not 
a substrate for aromatic amino acid decarboxy- 
lase but along with isotryptophan was a weak 
inhibitor. N-Acetyl-isotryptophan was a com- 
petitive inhibitor of chymotrypsin. 2,3-Dihy- 
drotryptamine was converted in vivo to 2,3-di- 
hydroindoleacetic acid probably via monoamine 
oxidase. 

In order to facilitate assays of the important 
enzymes, dopamine-/?-oxidase and tryptophan 
hydroxylase, methods were developed for 
preparation of 5-tritiotryptophan and /3,^-ditri- 
tiotyramine. These compounds are now being 
used to study the distribution and isolation of 
these enzymes (B. Witkop, J. Daly, Y. Fujita, 
K. Takase, A. B. Mauger and C. R. Creveling) . 

Methyltransferases. The use of catechol-0- 
methyltransf erase and radioactive S-adenosyl- 
methionine has been developed as a useful tool 
for studying the formation of a variety of cate- 
chols from simple phenols. An enzyme which 
hydrolyzes S-adenosylmethionine to S-adeno- 
sylhomocysteine and methanol has been charac- 
terized and has been found to be quite wide- 
spread in mammalian tissues. Knowledge of this 
enzyme's abundance in pituitary gland allowed 
a chemical classification of tumors of this or- 
gan. An enzyme which methylates phenol to 
anisol has been studied. This is one of the few 
known cases of methylation of a simple phenol 
in a mammalian system. The enzyme catechol- 
0-methyltransferase has been extensively pu- 



202 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



rifled for studies on methylation of catechols 
(J. Daly, B. Witkop and S. Senoh) . 

Novel Hydroxyamino Acids. y-Hydroxy-L- 
lysine prepared from L-lysine by photochemi- 
cal chlorination and subsequent reaction with 
silver acetate was cyclized with nitrosyl chlo- 
ride to a mixture of 80% czs-4-hydroxy-L-pipe- 
colic acid and 20% fmns-4-hydroxy-D-pipecolic 
acid. This established the f/ireo-configuration 
for y-hydroxy-L-lysine, which was converted to 
i/ireo-y-hydroxy-L-homoarginine, identical in 
all respects with the naturally-occurring amino 
acids from Lathyrus seeds (Y. Fujita, B. 
Witkop, and N. Izumiya). 

Rearrangement of Isocyano-peptides. De- 
hydration of gramicidin A (a linear N-formyl- 
pentadecapeptide ethanolamide) with a large 
excess ('—40 moles) of tosyl chloride in pyri- 
dine led to the unstable isocyanide. Compara- 
tively stable isocyanides were obtained by dehy- 
dration of N-formyl-DL-valine methyl ester 
and ethyl N-formyl-DL-valyl-glycinate. By con- 
trast N-formyl-DL-valine amine on dehydra- 
tion gave a-formylaminoisovaleronitrile, and 
N-formyl-DL-valyl-glycine amide gave N-for- 
myl-DL-valine cyanomethylamide via the ini- 
tially-formed unstable isocyanide by a rear- 
rangement which is pictured as an intramolec- 
ular dehydration (F. Sakiyama and B. Witkop) . 

Proline Analogs and Their Effects Upon 
THE Biosynthesis of Actinomycins. Synthetic 
3-methylproline has been separated into cis and 
trans isomers by several methods, and these 
isomers have been identified by steric hindrance 
studies. Derivatives of 3-methyl-4,5-dehydro- 
proline have been synthesized, cis- and trans-S- 
Methylproline are both potent inhibitors of 
actinomycin biosynthesis; 4-methylproline is 
less inhibitory and is incorporated into the 
actinomycin peptides in place of proline. Triti- 
ated actinomycin acid IV was prepared from 
tritiated actinomycin IV which had been pro- 
duced biosynthetically; its enzymic lactoniza- 
tion was not observed. A novel route was used 
to prepare 2,3-dihydrotryptophan labeled in the 
2- and 3-positions, which is required for meta- 
bolic studies (A. B. Mauger and B. Witkop). 

Reductive Photochemistry of Free and 
Bound (Aromatic) Amino Acids. The three 



aromatic amino acids undergo extensive reac- 
tions when irradiated with uv-light of varying 
wave-lengths in the presence of NaBHi. The 
reactivity under photoreductive conditions as 
well as the products formed vary in a charac- 
teristic way for each amino acid. Tryptophan, 
histidine and tyrosine undergo partial reduction 
of the aromatic portion of the molecule. From 
the photolysis solution of tryptophan 2,3- and 
4,7-dihydro-tryptophan were isolated. N-Acetyl- 
tryptophanamide is partially cleaved to 
indolepropionic acid, whereas N-chloro-acetyl- 
tryptophan yields N-acetyl-tryptophan and 
/?-indolylpyroglutamic acid under photoreduc- 
tive conditions. Imidazoline and imidazolidine 
structures have been assigned to the products 
obtained from histidine and its derivatives. The 
question of dimerization of the reduced histi- 
dine is presently under investigation (B. Wit- 
kop, F. Sakiyama, 0. Yonemitsu, J. Moore, 
T. Spande and P. Cerutti) . 

Reductive Photochemistry of Free and 
Bound Nucleotides. In the photo excited 
state uridine and its phosphorylated deriva- 
tives are reduced selectively in the presence of 
NaBHi to their 5,6-dihydro derivatives, where- 
as none of the other major nucleosides (-tides) 
are reduced. Dihydrouridylic acid and dihy- 
drouridine-5'-phosphate were synthesized on a 
preparative scale by this new method. The 
applicability to polynucleotides was demon- 
strated with poly U, yeast RNA and s-RNA 
from different sources. The specific loss of re- 
coverable uridylic acid and the formation of 
dihydrouridylic acid have been shown to be a 
function of exposure to photoreductive condi- 
tions. This new method for the synthesis of 
oligo- and polyneucleotides containing dihydro- 
uridylic acid residues permits the study of the 
function of these naturally-occurring residues 
in biogenetics, mechanism of base pairing, 
selective modification of triplets and mutagenic 
experiments by reductive flash photolysis 
(P. Cerutti, K. Ikeda, G. Balle and B. Witkop). 

Electrolytic Cleavage of Tyrosine Pep- 
tide Bonds and Effects of Tertiary Struct- 
ure. The cleavage of tyrosyl-peptide bonds by 
electrolytic oxidation has been applied to vaso- 
pressin and angiotensin, in each case showing 
total selectivity. In the case of ribonuclease, ox- 



I«H^ 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



203 



idation is incomplete, suggesting that selective 
attack at surface sites may be feasible. Under 
the reaction conditions, cystine is partially oxi- 
dized to cysteic acid (L. A. Cohen and L. 
Farber) . 

Mechanism of Oxidation of Tocopherol 
AND Ubiquinone. The oxidation of tocopher- 
ol by peroxides has been shown to proceed by 
an ionic rather than a radical mechanism. The 
pathway followed in phenol oxidations, in gen- 
eral, is determined, not by the valence change 
of the oxidant, but by its potential, in addition 
to other experimental factors (M. Oxman) . 

Catalysts of Ester Hydrolysis as Enzyme 
Models. The anion of N-acetylcycloserine, be- 
ing an 'Vnucleophile," is a potent catalyst for 
the hydrolysis of 2?-nitrophenyl acetate at neu- 
tral pH. Hydrolysis occurs by acetyl transfer, 
the acetyl group being accepted by the nitrogen 
atom of the heterocyclic ring (G. Milne) . 

Section on Analytical Services and Instrumenta- 
tion 

Approximately 10,500 chemical and instru- 
mental analyses were performed for 150 re- 
search scientists of NIH and for several scien- 
tists in other government agencies. Of this 
number many were non-routine determinations 
which required a varying amount of laboratory 
and literature research. While the total number 
of analyses performed has declined somewhat 
over the past several years the work load re- 
mains about the same owing to the wider va- 
riety of more complex analytical services being 
rendered. Special assistance was given to many 
research workers in the solution of non-routine 
analytical problems (W. C. Alford and H. K. 
Miller) . 

Section on Steroids 

Chemical Structure of Tomatillidine. 
Wolff-Kishner reduction of tomatillidine af- 
forded deoxotomatillidine (H) and catalytic 
reduction of the latter gave 5,6-dihydrodeoxoto- 
matillidine (HI). Treatment of HI with acetic 
anhydride yielded the 0,N-diacetyl-A"-dihydro- 
deoxo derivative (IV) which by acid hydrolysis 
was readily converted to 3/8-acetoxy-26-acetyl- 



aminocholestan-22-one (V) . V was oxidized by 
chromic acid into 3/3-acetoxybisnorallocholanic 
acid. These conversions established the struc- 
ture of deoxotomatillidine (II) as 25 (R) -22,26- 
imino-5 ,22 ( N ) -cholestadien-3j8-ol. This was 
confirmed by comparison with an authentic syn- 
thetic specimen. Spectral data (U.V., IR, NMR, 
mass spectrometry) indicated that the carbonyl 
function was located on C-24. The structure of 
tomatillidine is therefore (25R)-22,26-imino- 
5,22(N)-cholestadien-24-one-3/3-ol (Y. Sato, C. 
Djerassi, E. Bianchi, and H. Budzikiewicz) . 

Sterols from Parasites. In Taenia taenia- 
formis (cat tapeworm) cholesterol exists pre- 
dominantly as free cholesterol with only traces 
occurring as cholesterol esters. Sterols of Tryp- 
anosoma ranarum are being studied (Y. Sato 
with Theodor von Brand) . 

Preparation of Solasodine Glucoside. The 
monoglucoside of solasodine was prepared by the 
interaction of solasodine with l-bromo-2,3,4,6- 
tetracetyl-a-d-glucose through the Konigs-Knorr 
synthesis and by hydrolysis of the resulting 
tetraacetyl derivative with barium methoxide. 
Chromatography over alumina afforded a fairly 
pure product which was submitted for testing 
against cancer. A number of other glucosides 
of steroidal alkaloids are being studied (Y. 
Sato) . 

Alkaloids from Solanum Congestiflorum 
(Natri). Solanum congestiflorum (natri) a 
plant of South American origin affords four 
closely related steroidal alkaloids. The main 
component has been identified as (25R) -22,26- 
imino-5a-cholest-22(N)-en-3j8-ol, the structure 
of which has been established unambiguously. 
The others present in small quantities are sus- 
pected to be the epimeric hydroxy and oxo de- 
rivatives (Yoshihiro Sato and Yoshio Sato). 

Synthesis of Heterocyclic Steroids. The 
synthesis of 3'i-methylisoxazolino-[17,16-d]-5- 
androsten-3j8-ol has been realized in high yields 
(85-90%) by an elegant one step procedure 
involving oximation and concomitant ring 
closure of 16Qf-chloro-5-pregnen-20-one-3;8-ol 
acetate. It consists of treating the readily avail- 
able 16-chloropregnenolone acetate with hydrox- 
ylamine hydrochloride in pyridine at 105° for 
24 hours (Yoshio Sato and H. Kaneko). 



204 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Photochemical Reactions of Steroidal 
Alkaloids. The irradiation of N-nitrosatomat- 
idine in ethanol yields tomatidine but N-nitroso- 
solasodine fails to give solasodine. Instead, 
a new as yet unidentified substance is obtained. 
Irradiation of pseudotomatidine B in cyclo- 
hexane affords one product while in alcohol a 
number of substances are obtained (Yoshihiro 
Sato and Yoshio Sato) . 

Assay of Dopamine-/3-Oxidase Activity 
IN Sympathetic Tissue. A micro method 
for the determination of dopamine-/3-oxidase 
activity has been developed based on the re- 
lease of tritium labeled water from the enzyme 
substrate during the reaction. This assay has 
permitted the localization of this enzyme in var- 
ious tissue species and within sympathetic 
nerves (C. R. Creveling and J. Daly, cooperat- 
ing unit: Morton Levitt (LCB) ) . 

Microbiological Hydroxylation of Steroi- 
dal Alkaloids and Sapogenins. An enzymatic 
system capable of catalyzing the hydroxylation 
of the sapogenin, solasodine, to form 7j8-hydrox- 
ysolasodine has been isolated from the mold, 
Helicostylum piriforme (C. R. Creveling and Y. 
Sato). 

Metabolism of C"-Labeled Steriods by 
Adrenals from Rats with a Mammotropic 
Pituitary Tumor. The adrenal glands of rats 
bearing a transplantable mammotropic pitu- 
itary tumor are enlarged up to 10 times normal 
size due to very high levels of ACTH. In vitro 
incubation of this adrenal tissue with proges- 
terone-4-C" or deoxycorticosterone acetate-2-C" 
results in low yields of corticosterone and 
aldosterone with an accompanying increased 
production of two metabolites tentatively identi- 
fied as 18-hydroxy derivatives of deoxycorti- 
costerone and corticosterone. Present data sug- 
gests that the normal metabolic pathway of 
progesterone in this tissue is bypassed in favor 
of these 18-hydroxy compounds (D. F. Johnson; 
R.W.Bates (LNE)). 

Adrenal Metabolism of the Mongolian 
Gerbil. Chromatographic analysis of pooled 
blood samples indicate little or no circulating 
free adrenal steroids in this animal. Prelimi- 
nary results of in vitro incubations of adrenal 
tissue with progesterone-4-Ci' indicate a dif- 
ferent steroid pattern compared to that of the 
rat. Investigations are being carried out to de- 



termine any possible relationship between the 
steroid metabolism of the Gerbil and the low 
water loss observed in the animal (D. F. John- 
son and cooperating unit: H. Y. C. Wong, How- 
ard University). 

Programmed Gradient Elution Chroma- 
tography of Steroids. A flexible and efficient 
method of producing concentration gradients 
for column chromatography of steriod mixtures 
using a punch-tape mechanism was described 
earlier. The utility of this system has been in- 
creased by addition of a Friden Auxiliary Tape 
Punch Console, which allows the investigator to 
change programs as the elution process pro- 
gresses, rather than using pre-punched tapes 
(D.F.Johnson). 

Sterol Biosynthesis in Plants. Meva- 
lonic acid-2-C" and methionine-methyl-C" 
were incubated with leaf cuttings from soy- 
bean seedlings in an aqueous medium. Radioac- 
tive transformation products were isolated by 
thin layer chromatography. The presence of 
stigmasterol and /3-sitosterol has been estab- 
lished, and microchemical data indicates that 
at least two other radioactive metabolites are 
related stands. The results obtained suggest a 
precursor relationship between these completed 
structure sterols via hydrogenase and/or dehy- 
drogenase enzyme systems (D. F. Johnson and 
J. A. Waters). 

Anodic Decarboxylation of Carboxylic 
Acids. Various glycidic acids are being oxi- 
dized in methanol at a platinum anode to study 
the mechanism of decarboxylation with refer- 
ence to the opening of the epoxide group. The 
two major products (of,^-unsaturated ketone 
and methoxy ketone) suggest ring opening via 
carbonium ion intermediates from two possible 
pathways (J. A. Waters). 

LABORATORY OF MOLECULAR BIOLOGY 

Relation Between Structure and Function of 
Clutamate Dehydrogenase from Bovine 
Liver 

1. Chemical structure of the subunits of 
GDH — The N-terminal amino acid of gluta- 
mate dehydrogenase is L-alanine. The C-ter- 
minal amino acid is L-threonine. Hydrolysis by 
trypsin or cleavage of the peptide chains by 
cyanogen bromide indicates that glutamate de- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



205 



hydrogenase is composed of a single type of sub- 
unit with a molecular weight of 50,000. 

2. Molecular weight of enzymically active 
units — The polymeric form of glutamate dehy- 
drogenase which exists above 2 mg per ml in 
solution has a molecular weight of 2,000,000 
and is highly asymmetric. The molecular 
weight of the enzymically active monomeric 
unit catalytically active for glutamate and 
alanine is about 400,000 and may, under ex- 
treme circumstances, disaggregate to forms of 
lower molecular weight. 

3. Immunological studies — A theory has 
been proposed to account for the separation of 
three immunologically different forms of the 
enzyme in rapid equilibrium. In addition anti« 
genie relations between glutamate dehydrogen- 
ases from different organs of beef, different 
mammalian sources, and microorganisms have 
been studied. The "alanine form" of glutamate 
dehydrogenase has been found to be catalyti- 
cally active for other monocarboxylic L-amino 
acids such as leucine, isoleucine, methionine 
and valine as well as the amino acid analogs, 
norvaline and norleucine. The "glutamate 
form" appears to be active only for L-gluta- 
mate and not for other dicarboxylic amino 
acids. 

4. Allosteric effects — The allosteric effects 
observed with the glutamate dehydrogenase 
system appear to be only noncooperative. These 
findings have theoretical implications for the 
kinetic model of allosteric interactions recently 
developed. (G. Tomkins, E. Appella, K. L. 
Yielding, M. W. Bitensky, N. Talal). 

Enzyme Induction in Bacteria 

Kinetic studies indicate that yS-galactosidase 
is induced prior to thiogalactoside transacety- 
lase on enzyme in the same operon. On deinduc- 
tion j8-galactosidase synthesis stops before that 
of the acetylase. These results have been inter- 
preted in terms of the direction of reading the 
messenger RNA of the lactose operon. (G. 
Tomkins, D. Alpers) 

Enzyme Induction in Mammalian Cells 

Studies of hydrocortisone induced trypto- 
phan pyrrolase and tyrosine transaminase indi- 
cate that partial regulation of the synthesis of 



these enzymes occurs at the level of messenger 
RNA translation rather than transcription. 
Additional studies indicate that the repressor 
operating at the translational level has a rapid 
rate of turnover, depends on RNA synthesis 
and is probably a protein. Complementary stud- 
ies indicate that ribosomes in liver cytoplasm 
primarily exist attached to membranes in the 
form of polyribosomes and that a small number 
are unattached to the membrane. No intrinsic 
difference between these ribosomes was found 
nor was their rate of synthesis different. The 
turnover time of ribosomal RNA has been esti- 
mated to be 5 days. (G. Tomkins, John Loeb, B.- 
Peterkofsky, S. Hayashi, L. Garren) 

Serine Transacetylase 

An enzyme has been isolated which transfers 
the acetyl moiety from acetyl coenzyme A to 
the amino group of L-serine. The enzyme has 
been partially purified and some of its charac- 
teristics have been studied. In addition, acetyl 
serine has been identified as a component of 
certain proteins in the cytoplasm of E. coli. (G. 
Tomkins, N. Kredich) 

Mechanism of Action of Steroid Hormones 

The major portion of these studies has been 
done on minimal deviation hepatomas carried 
in tissue culture. Steroid hormones were ob- 
served to induce tyrosine a-ketoglutarate trans- 
aminase in the cultured cells. The kinetics of 
induction, dose-response curves, media require- 
ments, and response to inhibitors of various 
types were then studied. The morphology of 
the cells was studied by light and electron mi- 
croscopy. (E. Brad Thompson) 

Active Transport of Amino Acids in Salmonella 
Typhimurium 

Further work has been done on the transport 
mechanism for amino acids. The formation of 
intermediates during active transport, between 
lipids and amino acids has been demonstrated. 
The nature of these intermediates is such to 
justify a possible role in active transport, i.e., 
the amino acid is unchanged and can be sepa- 
rated from the lipid by very mild treatment 
with alkali ; the intermediates are formed very 



206 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



rapidly in very small amounts and they are lip- 
id soluble. There is a suggestion that each 
might correspond to a phospholipid with a spe- 
cific conformation of fatty acids. 

The whole lipid fraction and the amino acid 
intermediates are under further investigation 
in order to characterize better their structures 
and roles. Mutants missing various permeases 
will be used in the process. (Giovanna Ames) 

Amino Acid Incorporation into Protein in Cell 
Free Systems: Influence of Ordered Struc- 
tures Between Purine Ribosides and Poly- 
ribonucleotides on Messenger Activity 

Monomeric nucleic acid components (nucleo- 
tides, nucleosides or purines) are capable of 
forming regular ordered structures with com- 
plementary polyribonucleotides (Miles, How- 
ard and Frazier). These observations sug- 
gested that monomer-polymer interactions 
might be involved in some biological control 
mechanisms, since the secondary structure of 
nucleic acids is important for their function. 
To test this possibility we examined the effect 
of purine ribosides on polyribonucleotide di- 
rected amino acid incorporation in cell free 
systems from rat liver and E. coli. The ability 
of poly U to function as messenger RNA can be 
reduced or abolished by complementary ribo- 
sides (adenosine or 2-6 diamino purine). The 
degree of inhibition is a function of tempera- 
ture and of concentration of riboside. Qualita- 
tively good correlations are observed between 
conditions favoring inhibition in the biological 
system and those required for the formation of 
helices as measured by physical chemical meth- 
ods. The site of action of the ribosides was de- 
lineated by examining their influence on the 
binding of poly U to ribosomes and by studying 
their effect on the rate of degradation of poly 
U in the systems. The ribosides exert dual 
effects upon poly U. They prolong its lifetime 
in the system and, at the same time, by de- 
creasing its binding to ribosomes they suppress 
its activity as messenger UNA. Preliminary ex- 
periments with a copolymer of UC indicate 
that ribosides of complementary bases can in- 
fluence amino incorporation directed by copoly- 
mers. When amino acid incorporation is di- 
rected by endogenous natural messenger RNA, 



small temperature dependent inhibitions by pu- 
rine ribosides are observed. (E. S. Maxwell, H. 
T. Miles, F. B. Howard) 

Helix Formation Between Polynucleotides and 
Purine Monomers 

Purines, purine nucleosides and purine nu- 
cleotides of appropriate structure interact with 
polynucleotides to form two or three-stranded 
helices analogous to those formed between 
pairs of polynucleotides. Base pairing specific- 
ity is observed in these interactions. Pyrimi- 
dines and their derivatives appear to be unable 
to serve as monomer components in such inter- 
actions. The phenomenon has provided a con- 
venient model system for the study of factors 
affecting nucleic acid stability and may be of 
importance in the control of some biologically 
important reactions. The method of infrared 
spectroscopy in aqueous solutions was further 
developed to study the formation and dissocia- 
tion of these complexes. (H. T. Miles, F. B. 
Howard; biochemical aspects of the problem 
were studied in collaboration with E. Maxwell 
and M. F. Singer) 

Chemistry of Isoguanosine 

Isoguanosine in aqueous solution forms a 
regular ordered structure which is highly 
asymmetric and presumably helical. The 
phenomenon was studied by infrared and ultra- 
violet spectroscopy, optical rotation, and con- 
centration dependence of viscosity. The tauto- 
meric structure and site of protonation of the 
nucleoside have been studied by infrared and 
nuclear magnetic resonance spectroscopy. 

The 5'-phosphate of isoguanosine has been 
synthesized by the cyanoethylphosphate meth- 
od, and preliminary experiments indicate that 
it does not undergo self-structure formation. 
(R. V. Ravindranathan) 

Optical Properties of Nucleic Acid 

We have shown that for a random sequence 
of bases, it will be true quite generally that the 
hyperchromism of DNA at a given wavelength 
will be a quadratic function of the base compo- 
sition of the region undergoing denaturation. 
Thus, three parameters at each wavelength 



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NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



207 



suffice to predict the contribution to hyperchro- 
mism of a region of known base composition. 
We have obtained the parameters at each wave- 
length by measurement of a large number of 
bacterial DNA hyperchromic spectra. It is pos- 
sible to use the equations to determine the com- 
position of each region of a denaturing DNA 
molecule as it denatures, and thus to obtain a 
"map" of the DNA. Important evidence con- 
cerning the structure of viral DNA's and par- 
ticularly of ADNA, has been obtained by this 
method. 

We have also shown that the helix and coil 
spectra of DNA can be analyzed separately in a 
manner similar to that used for hyperchro- 
mism. The method presently is sufficiently pre- 
cise to permit accurate determination of the 
base composition and concentration of an un- 
known DNA from its native or high-tempera- 
ture spectra alone. If both spectra are known 
an accurate determination of the amount of 
denatured material present in the original 
sample can also be made. (Gary Felsenfeld, 
Shalom Z. Hirschman) 

Polyadenylic Acid at Ph 7 

It has already been shown in this laboratory 
that the neutral poly A structure undergoes 
thermal disruption by a completely non-cooper- 
ative process, so that the process can be ana- 
lyzed well by means of the van't Hoff method, 
which yields an "apparent AH" for the reac- 
tion. We have accurately redetermined the val- 
ue of aH for poly A, and compared it with 
that for the oligonucleotide series. Aside from 
what appear to be polyelectrolyte swelling con- 
tributions, values of aH for all members of 
the series are the same. It is possible to con- 
clude that poly A is composed of small inde- 
pendently denaturing units, about two nucleo- 
tides long each, and that there is no long 
range ordered, single stranded structure. ( Marc 
Leng, Gary Felsenfeld) 

Internal Proteins of Bacteriophage T2 

Other workers have demonstrated that at 
least two proteins are released from bacterio- 
phage T2 by osmotic shock (e.g., Hershey, 
A. D., Virology 1: 108 (1955); ibid., A: 237 



(1957) ). The proteins are distinguishable by 
their solubility in cold trichloracetic acid. 

Current work has separated these compo- 
nents on acrylamide P-60 columns. The acid-sol- 
uble protein has also been chromatographed as 
a single peak on DEAE-cellulose. The acid-insol- 
uble protein has resisted further purification 
procedures, largely due to irreversible surface 
adsorption on most materials. 

Pulse-labelling of T2-infected E. coli indi- 
cates both proteins are made approximately 
coordinately during infection, their synthesis 
beginning within three minutes after infection 
starts. (Steven B. Zimmerman, Gary Felsen-, 
feld) 

Ribonuclease Activity of Pancreatic Deoxyri- 
bonuclease 

It was previously found that a small but sig- 
nificant RNase activity persisted in pancreatic 
DNase through various chromatographic 
procedures. Further purification attempts 
failed to separate the two activities. However, 
it was possible to inactivate differentially the 
RNase or the DNase. N-Bromosuccinimide 
completely destroyed DNase activity without 
altering RNase levels; sodium iodoacetate re- 
duced RNase activity to levels indetectable by 
the ultrasensitive assay while recovery of 
DNase was 70-90%. (Steven Zimmerman) 

Viscosity Studies of DNA 

High precision viscosimetric study of bacte- 
riophage DNA. 

The intrinsic viscosity of DNA (sahnon 
sperm, calf thymus T4 bacteriophage) in the 
presence of uni-univalent electrolytes decreases 
in a continuous manner as the ionic strength is 
increased and then attains a limiting value at 
about 0.5 M. 

The concentration dependence of the reduced 
viscosity may be abolished and regenerated by 
the introduction and removal of trace amounts 
of basic proteins, polypeptides and polyamines. 

In contrast to earlier reports the marked 
sensitivity of the intrinsic viscosity of the 
DNA molecule to its ionic environment repre- 
sents typical polyelectrolyte behavior. The vis- 
cosity behavior observed with the deliberately 



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ANNUAL REVIEW OP INTRAMURAL RESEARCH 



contaminated DNA samples resembles most of 
that reported in earlier viscosity studies, indi- 
cating that a large body of previous work on 
DNA probably was performed on impure ma- 
terial. (Philip D. Ross) 

Three-Stranded Poly (A-f-2U) Complex 

Calorimetric measurements of the heat of 
the addition of the second strand of poly U to 
poly (A+U) to form the three-stranded poly 
(A+2U) complex in 0.1 M and 0.5 M NaCl at 
24° and 37° are reported. A value of 
aH=— 3800 cal. (mole of poly (A-}-2U) 
formed)-! was found to be fairly insensitive 
to the experimental conditions employed. The 
heat of the addition of the third strand to the 
preformed poly (A+U) helix is considerably 
less exothermic than the heat of reaction be- 
tween poly A and poly U to form poly (A -I- U) . 
The insensitivity of the heat of additon of the 
third strand (poly U) to changes in salt con- 
centration and temperature lends qualitative 
support to the earlier hypothesis that the ma- 
jor portion of the variation of the AH of the 
poly A and poly U reaction with salt concentra- 
tion and temperature arises from differences in 
the conformation of poly A. 

Combining the information obtained in this 
study for the AH of the reaction forming poly 
(A+2U) with previous data for the aH of the 
formation of poly (A-f-U) indicates that the 
conversion of poly (A + U) to poly (A + 2U) 
is opposed by an enthalpy change which in- 
creases with temperature. Extrapolation of 
these values to 52° where poly (A + U) is 
transformed to poly (A + 2U), in 0.5 M NaCl 
leads to a value of aH= +3800 cal. (mole (A 
+ 2U) formed)-^ and AS = 11.5 cal. (mole of 
(A+2U) formed -^ deg. -\ The latter value 
of 11.5 entropy units derived from the experi- 
mental data may provide a good estimate for 
the entropy change of a single random coiled 
polynucleotide entering a helical structure. Sim- 
ilar values have been previously found for 
poly (A+U) and DNA. It is concluded from 
the calorimetric data that the driving force for 
the interesting poly (A+U) to poly (A+2U) 
conversion reaction must be the favorable en- 
tropy change. (Philip D. Ross) 



Protein Structure 

A correlation has been demonstrated be- 
tween the aplha helix content of globular pro- 
teins as determined by X-ray diffraction and 
optical rotatory dispersion measurements and 
their amino acid composition. Specifically, 
there appears to be a linear relationship be- 
tween the helix content of a variety of proteins 
and their percent composition of the amino 
acids, serine, threonine, isoleucine, valine, cys- 
teine and proline. (David R. Davies) 

Structural Studies on Nucleic Acids, Polynucleo- 
tides and Nucleotides 

The interaction of dyes of the acridine class, 
such as proflavine and acridine orange with 
DNA have been investigated. It has been 
shown that under conditions of high hydration, 
diffraction patterns can be obtained from fibers 
of dye-DNA complexes which show marked 
changes from the diffraction patterns of so- 
dium-DNA fibers. These changes consist of an 
increase in the layer line spacing and a dis- 
order of the diffraction pattern. 

A study of the optical transforms of various 
simple models has shown that there are two 
which are consistent with this type of change 
in the diffraction pattern. In one of these the 
dye is intercalated between the base pairs of 
the DNA, and in the other the dye binds on the 
outside of the DNA and produces large 
changes in the helical screw of the DNA. Tak- 
en in conjunction with measurements on the 
increases in length of the fibers as a function 
of the layer line spacing and with the observa- 
tions of other workers, these results have been 
interpreted as providing support for the inter- 
calation hypothesis. (David R. Davies and Da- 
vid Neville) 

Stable Heterogenotes of Bacteriophage T4 

T4 phages carrying two copies of the rll 
region have been described (see 1964 Annual 
Report) . Our recent work has been directed at 
understanding the nature of this genetic anom- 
aly. A series of three-factor crosses, with 
genetic markers straddling the rll region, has 
shown that the two rll genes are closely linked. 
The structure may thus be similar to that of 
genetic duplications in higher organisms, and 



NATIONAL INSTITUTE OP ARTHRITIS AND METABOLIC DISEASES 



209 



is definitely unlike that of normal (unstable) 
T4 heterozygotes. 

Recent genetic evidence (Weil et al.) sug- 
gesting that the duplication may extend well 
beyond the rll region is in course of investiga- 
tion by physical chemical methods in order to 
see whether there is a correlation between the 
size of the DNA of the mutant phage and the 
extent of the duplication. (Martin Gellert, Phil- 
ip D. Ross) 

Biochemical Control Mechanisms in Histidine 
Biosynthesis 

Further work has been done on the interme- 
diates, enzymes, genes and control mechanisms 
of histidine biosynthesis. 

1. A new intermediate in the pathway of 
histidine biosynthesis has been isolated, and 
shown to have the structure phosphoribosyl- 
AMP. Two enzymes have been described: PR- 
ATP-pyrophosphohydrolase, Avhich forms PR- 
AMP from PR-ATP, the first intermediate of 
the pathway, and PR-AMP, 1,6-cyclohydrolase, 
which opens the purine ring of PR-AMP, con- 
verting it to the next intermediate of the path- 
way. (D. W. E. Smith, B. N. Ames) 

2. Two enzymes of histidine biosynthesis 
have been purified from Salmonella: the first 
enzyme of the pathway and lAP-transaminase. 
The transaminase is a single chain of 67,000 
MW. with a C-terminal valine and an N-ter- 
minal serine. The pure enzymes are necessary 
in the study of the biochemical basis of polar- 
ity, modulation, feedback inhibition, and re- 
pression. (R. G. Martin, M. J. Voll, E. Appella, 
B. Ames) 

3. An extensive study of polarity mutants in 
the transaminase gene indicates that most mu- 
tants are polarity mutants. The theory of mod- 
ulation and polarity has been developed further. 
(R. G. Martin) 

4. The important pool in repression of the 
histidine biosynthetic enzyme appears to be that 
of histidine-sRNA rather than that of histidine. 
This conclusion is based on the isolation of 
various mutants with decreased histidine acti- 
vating enzyme that accumulate histidine yet 
are derepressed for the histidine biosynthetic 
enzymes. (John Roth and P. E. Hartman of 
John Hopkins, and B. Ames) 



5. One aspect of the mode of action of the 
widely used herbicide amitrole (aminotriazole) 
has been determined. The compound inhibits 
imidazole glycerol phosphate dehydrase, one of 
the enzymes of histidine biosynthesis. (J. L. 
Hilton, P. C. Kearney of U.S.D.A., and B. 
Ames) 

Chemistry and Genetics of Hemoglobin and 
Other Proteins 

In order to study the forces that hold the sub- 
units of hemoglobin together, lysyl residues 
were converted to homoarginyl residues by 
conversion of £-amino groups to e-guanidino 
groups. Guanidinated human adult CO-hemo- 
globin has the absorption spectrum of untreat- 
ed hemoglobin and forms large prismatic 
crystals. Solutions of this derivative are 
considerably more resistant than unmodified 
hemoglobin both to denaturation and to disso- 
ciation of high pH. The structure of the addi- 
tion compound of benzil and arginine has been 
established. This chemical modification results 
in resistance of the arginyl site to tryptic 
hydrolysis. (H. A. Itano, A. J. Gottlieb, K. Toi) 

Mechanism of Lambda Bacteriophage Induction 

We are encouraged to believe, from a study 
of a process called "curing", that the viral 
functions involved in lysogenic induction may 
be similar or identical for bacteriophages dif- 
ferently repressed. In curing, a superinfecting 
phage appears to provide the necessary func- 
tions for excising prophage from some bacte- 
rial nuclei without irrevocably damaging either 
these nuclei or the cells in which they are locat- 
ed. Excision of prophage by a lytic superinfect- 
ing phage without concomitant expression of 
prophage functions can also be demonstrated. 

A search among mutants of the well-studied 
bacteriophage lambda, has revealed one which 
appears incapable of curing yet which retains 
its other functions. Once we are in a position to 
specify genetic blocks in functions unique to 
the induction process we are in a position to 
understand this remarkably precise rearrange- 
ment of genetic material. (M. Yarmolinsky) 

Host-Induced Modification 

An investigation has been made of the 
growth restriction of E. coli strains against the 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



host modified phage T*4 and also the permis- 
siveness of Shigella disentheriae strains (Sh) 
for the phage. 

On the basis of previous results, it has been 
postulated that an RNA-inhibited endonuclease 
is responsible for restricting ability of E. coli 
B against T*4, containing nonglucosylated 
DNA. This hypothesis is now under test (T. 
Fukasawa) 

CUNICAL INVESTIGATIONS 

INTRODUCTION 

The clinical research program of NIAMD re- 
ceived a severe setback at the outset of this fis- 
cal year in the loss of Dr. Joseph J. Bunim, 
who had so ably supervised clinical investiga- 
tions since before the Clinical Center was 
opened. His post as Chief of the Arthritis and 
Rheumatism Branch remains unfilled at this 
writing, but it is anticipated that the name of a 
successor will soon be formally announced. 

In September 1964, under the auspices of the 
American College of Physicians, the Arthritis 
and Rheumatism Branch, NIAMD, conducted a 
five-day course at the Clinical Center entitled 
"Current Clinical and Laboratory Investiga- 
tions in the Rheumatic Diseases." Planning for 
this teaching exercise had been initiated by Dr. 
Bunim, and the project was carried through by 
members of the staff. An outstanding group of 
19 guests and 11 NIH scientists served on the 
faculty. Fifty physicians representing all areas 
of the country were registered and participated 
in the course. 

In November 1964, NIAMD and the Clinical 
Center served as hosts for the American Rheu- 
matism Association interim scientific meeting. 
This tradition had been established some years 
ago by Dr. Bunim, and was carried on this 
year by other members of the staff. 

During the period that the Arthritis and 
Rheumatism Branch has been without a chief, 
a reduction in its clinical activity has been una- 
voidable. This fact, and the temporary absence 
of Dr. J. E. Seegmiller, have resulted in a tem- 
porary decrease in the Institute's patient-care 
activities. During the period March 1, 1964 to 
February 28, 1965, NIAMD admitted 508 pa- 
tients for a total of 18,593 hospital days. The 



average patient stay was 37 days, and the aver- 
age census 70% of capacity. During the same 
period, there were 2318 outpatient visits for 
study and/or treatment. 

ARTHRITIS AND RHEUMATISM BRANCH 
Clinical Observations on Rheumatic Diseases 

Association of Lymphoma and Sjogren's 
Syndrome. Through the years a number of 
patients with Sjogren's syndrome have been 
admitted to the Clinical Center for observation. 
It was noted that an unexpectedly high percent- 
age of these patients manifested lymphadeno- 
pathy with malignant degeneration. Four such 
cases were reported in 1964. Two of these pa- 
tients are still alive, and are under continuing 
study at the Clinical Center. During the past 
new cases were seen, in one of whom a biopsy 
showed benign lymphatic hypertrophy. The 
other was diagnosed as lymphoma. In addition, 
pathologic material on two similar cases has 
been obtained from other institutions for com- 
parative study. This descriptive study is of 
particular interest because of the possible in- 
volvement of auto-immune mechanisms in the 
etiology of lymphoma on the one hand, and of 
Sjogren's syndrome on the other. (Dr. Talal) 

Clinical Observations on Juvenile 
Rheumatoid Arthritis. In the decade since 
the Clinical Center opened, 60 patients with ju- 
venile rheumatoid arthritis have been ad- 
mitted. These medical records have all been re- 
viewed, and as many of the patients as possible 
have been reexamined. The data on the natural 
history of juvenile rheumatoid arthritis, as ex- 
emplified by this experience, are being collected 
and analyzed in the light of a review of the ex- 
isting literature on the subject. It is hoped that 
this study will provide useful background ma- 
terial and guidance for future clinical investi- 
gation of the syndrome. (Drs. Washburn and 
Alepa) 

Chronic Effects of Allopurionl on 
Gout. Allopurinol is effective as an inhibitor 
of xanthine oxidase, and is now available for 
experimental use in the treatment of gout. Its 
effect is to decrease the conversion of xanthine 
and hypoxanthine to uric acid, so that blood 



' 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



211 



urate levels fall and xanthine and hypoxanthine 
levels rise. It offers promise as a drug for 
the management of gout if it can be well toler- 
ated over long periods. In the present study, 
four patients with chronic gout, three of whom 
had measurable tophi, were started on 400 mil- 
ligrams of allopurinol per day. So far, the drug 
has been well tolerated for periods up to a 
year, with an effective decrease in plasma 
urate concentrations. Xanthine excretion in the 
urine increased, but with the maintenance of 
an adequate fluid intake, there was no evidence 
of renal stone formation. The patients are still 
on treatment and under observation; if the 
promising results of the study to date continue 
to apply in the future, it would appear that this 
drug will have therapeutic potential in those 
patients who for one reason or another cannot 
be properly managed with uricosuric agents. 
(Drs. Alepa and Seegmiller.) 

Studies in Serology and Immunochemistry 

Antibodies to Human Gamma Globulin 
Among Patients with Rubella. Thanks to 
the cooperation of the Perinatal Research 
Branch, NINDB, paired serum samples were 
obtained on each of 53 patients infected during 
a recent rubella outbreak on an Arctic island. 
These sera have been examined with the ben- 
tonite fiocculation test in order to evaluate 
further the significance of reports indicating 
the presence of rheumatoid factor in patients 
with rubella complicated by arthritis. While 
low titers of rheumatoid factor were found in 7 
of the 53 initial serum samples, higher titers 
were present in 39 of the 53 specimens taken 
five months later. This represented an increase 
in titer of 3 tubes or more in 69% of the 
samples. These results indicate that an acute 
self-limited viral infection, like chronic infec- 
tions in man, can give rise to positive bentonite 
fiocculation tests. (Drs. Alepa and Monif 
(NINDB).) 

Genetically Determined Gamma Globulin 
Polymorphism in Chimpanzees. The geneti- 
cally determined antigens (Gm and Inv) found 
in human gamma globulin have been useful 
markers in the study of immune mechanisms. 
These antigens can be recognized only by the 
use of specific antisera obtained from rare pa- 



tients who develop sensitization to these fac- 
tors. No animal sera with anti-Inv(b) speci- 
ficity have so far been prepared. Therefore, a 
study has been undertaken to determine the dis- 
tribution of Gm and Inv types among chimpan- 
zees, in hopes that by immunizing one animal 
with gamma globulin from another, antisera 
can be produced at will which will be of use in 
human genetic studies. (Dr. Alepa with Dr. 
Terry (NCI) and Dr. Moor-Jankowski (Emory 
Univ.) .) 

Interactions Between Polypeptide 
Chains of Gamma Globulin. Previous stud- 
ies in this and other laboratories have dem- 
onstrated that IgG antibody molecules can be 
separated in vitro into heavy and light poly- 
peptide chains. These chains can then be in- 
duced to recombine yielding an immunologi- 
cally active antibody molecule. It has thus been 
possible to investigate various factors, biologi- 
cal and chemical, which govern the makeup of 
naturally produced antibody molecules. In the 
present study fragments derived from the dis- 
sociation of nonspecific rabbit IgG protein was 
allowed to recombine with fragments derived 
from a specific antibody directed against the 
2,4-dinitrophenyl hapten. The polypeptide 
chains were separately identified by suitable la- 
beling with I"^ and P-^ It was found that 
in the absence of the DNP hapten, chance alone 
governed the recombination of heavy and light 
chains. In the presence of the hapten group, 
however, recombination of heavy and light 
chains from the specific antibody molecule was 
favored. These findings indicate that both the 
heavy and light chains probably participate in 
the formation of the specific immunologically 
active site, and may throw further light on the 
mechanism of induction of antibody synthesis 
in vivo. (Drs. Metzger and Mannik.) 

In a related study, a homogenous abnormal 
immunoglobulin was obtained by purification 
of human myeloma protein. Heavy and light 
chains from this protein were purified, and 
then allowed to recombine in the presence of 
polypeptide chains derived from other antibody 
molecules. By the use of I"^ and I"= labels 
to trace specific polypeptide chains, it was 
found that heavy chains tended to recombine 
with light chains derived from the same origi- 



212 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



nal antibody molecule. (Dr. Mannik with Dr. H. 
Gray (Rockefeller Inst.).) 

Characterization op a Human Macro- 
globulin. A macroglobulin (IgM) obtained 
from a patient was highly purified by gel filtra- 
tion. The molecular weight of this protein was 
found to be 892,000, and subunits formed by 
partial dissociation had a molecular weight of 
185,00. This is consistent with the view that 5 
subunits are polymerized to form one macro- 
globulin molecule. Subunits were dissociable 
into heavy and light chains, as are other im- 
munoglobulins, with a yield of two heavy and 
two light chains per subunit. The heavy chains 
derived from IgM were of greater molecular 
weight than those derived from IgG ; only part 
of the difference can be accounted for on the 
basis of carbohydrates. (Drs. Metzger and 
Miller.) 

Metabolism of Altered Immunoglobu- 
lins. Purified rabbit gamma globulin, after la- 
beling with I^^^, was readministered to rab- 
bits and its metabolism followed by standard 
methods. This experiment was done with unal- 
tered gamma globulin and with the protein 
after reduction and alkylation. The destruction 
of the disulphide bridges between polypeptide 
chains of these immunoglobulins did not cause 
the molecule to break up into fragments in 
vivo. Surprisingly enough, the altered mole- 
cule, which might have been expected to be less 
stable, had a longer half-life in the recipient 
animal's plasma than did the native antibody. 
(Drs. Cohen and Mannik.) 

Biochemical Studies 

Protein Synthesis in the Spleen. Sub- 
cellular fractions prepared by differential cen- 
trifugation of homogenates of rat splenic tis- 
sue are being studied with regard to their ac- 
tivity in protein synthesis. It is hoped that with 
further development, these methods will yield a 
cell-free preparation suitable for the investiga- 
tion of antibody synthesis in vitro. A polyribo- 
some preparation obtained from splenic tissue 
has been found to incorporate radioactivity 
from labeled amino acids. The reaction depends 
on magnesium and another soluble co-factor, 
and is inhibited by puromycin or by ribonu- 
clease. The nature of the newly-formed protein 



is not yet known, but experiments to study its 
relationship to gamma globulin are in progress. 
(Dr. Talal.) 

Immunochemical Study of Glutamic 
Dehydrogenase. Preparations of glutamic 
dehydrogenase from different sources have 
been tested immunochemically and electropho- 
retically for structural differences. The sources 
of glutamic dehydrogenase were six different 
bovine tissues, and one tissue, liver, from eight 
different vertebrate species. All preparations 
of beef glutamic dehydrogenase appeared to be 
identical. The dehydrogenase preparations 
from the livers of various species could be dis- 
tinguished, although in some cases no immuno- 
chemical difference could be shown. A prep- 
aration of glutamic dehydrogenase from a 
bacterial source, however, did not cross-react at 
all with these preparations. This study is of 
interest in relating biochemical differences to 
species which have developed during the course 
of evolution. (Drs. Talal and Tomkins (A- 
LMB).) 

Study of Human Lactate Dehydrogenase 
Isozyme Pattern. Preparations of lactate 
dehydrogenase prepared from the red blood 
cells of 1,200 hospital patients in the Washing- 
ton, D.C. area have been studied by starch gel 
electrophoresis. The cell donors included 600 
Negroes and 600 Whites. The electrophoretic 
mobilities of lactate dehydrogenase prepara- 
tions demonstrate a genetically controlled poly- 
morphism which can be analyzed in terms of 
two genetic loci. Electrophoretic patterns show 
up to five lactate dehydrogenase fractions, the 
relative quantities of which vary from one in- 
dividual to another; these patterns can be ex- 
plained on the basis of the formation of tet- 
ramers from monomeric units not all of which 
are identical. Dissociation of the subunits of 
lactate dehydrogenase can be accomplished in 
vitro, and recombinants prepared artifically. 
(Dr. Vesell.) 

Biochemistry of ACTH. The effect of 
ACTH on the adrenal gland of the intact rat is 
being investigated. Steroid secretion is meas- 
ured by the analysis of adrenal vein blood. Ra- 
dioisotope incorporation into the glandular tis- 
sue serves to measure the rates of protein and 
nucleic acid synthesis. Results to date show 
that agents which prevent protein synthesis, 



NATIONAL INSTITUTE OP ARTHRITIS AND METABOLIC DISEASES 



213 



such as cycloheximide and puromycin, prevent 
the effect of ACTH in stimulating steroid se- 
cretion. Inhibition of nucleic acid synthesis by 
actinomycin D does not have this effect. Con- 
tinuation of these investigations promises to 
throw further light on the intimate mecha- 
nisms by which polypeptide hormones can con- 
trol the cellular metabolism in the target or- 
gan. (Dr. Garren.) 

Hormonal Regulation of Enzyme In- 
duction. Hydrocortisone administered to rats 
causes an increase in the rate of synthesis of 
the liver enzymes, tryptophan pyrrolase and 
tyrosine transaminase. Since the stimulation of 
these enzymes occurs within 30 minutes after 
hormone administration, they have been stud- 
ied as a model of hormone regulation of pro- 
tein synthesis. 

Studies utilizing this system have indicated : 
1) that DNA (MRNA) dependent RNA syn- 
thesis is involved, since enzyme induction is 
prevented by actinomycin D which blocks RNA 
synthesis; 2) that the template RNA for these 
enzymes is stable, since non-induced enzyme 
synthesis continues for at least six hours in the 
presence of complete inhibition of further 
RNA synthesis. (If MRNA turned over rap- 
idly, as in bacteria, enzyme synthesis would 
cease within minutes); 3) that following en- 
zyme induction another regulatory mechanism 
is activated which inhibits further synthesis of 
these enzymes. This inhibition apparently does 
not involve the level of MRNA, which is appar- 
ently stable, but appears to operate directly at 
the level of the cellular protein synthetic ma- 
chinery (translation of MRNA). The abrupt 
decrease in the rate of enzyme synthesis which 
usually occurs four hours after hormonal stim- 
ulation can be prevented by administration 
of actinomycin D. This suggests that a DNA 
dependent RNA is made at this time which in- 
hibited further enzyme synthesis. The possibili- 
ties of this model system for investigating 
mechanisms of regulation of protein synthesis 
have not been exhausted. (Dr. Garren.) 

Biochemistry of Carbon-Fluorine Bond. 
A soil bacterium which can utilize fluoroace- 
tate as its sole source of carbon has been 
isolated. This organism possesses a defluorinat- 
ing enzyme which has been partially purified, 
and the mechanism of the reaction that it cata- 



lyzes is being studied. The fluorine is replaced 
by a hydroxyl group, with the liberation of 
fluoride ion. Fluoroacetate and iodoacetate are 
similarly attacked, although at a slower rate. 
The enzyme, however, does not attack the car- 
bon-fluorine bond in other compounds. Because 
of the increasing use of fiuorinated analogues 
of hormones and metabolites as therapeutic 
agents, further investigation of the biochem- 
istry of fluorine-carbon compounds may be of 
pharmacologic interest. (Dr. Goldman.) 

Biochemistry op Maple Syrup Urine 
Disease. The biochemical properties of the 
decarboxylase for the keto-acids derived from 
leucine, isoleucine, and valine are being investi- 
gated in tissues obtained from animal and hu- 
man sources, including fibroblasts cultured in 
vitro from skin explants of a patient with 
Maple Syrup Urine Disease. (Dr. Seegmiller 
with Drs. Westall and Dent, Univ. College Hos- 
pital, London.) 

Biochemistry op Cystinosis. The biochemi- 
cal abnormalities leading to the deposition of 
cystine crystals in the tissues of patients with 
cystinosis are being investigated by studies of 
the metabolism of the sulfur-containing amino 
acids both in vivo and in vitro. Comparisons 
will be made of the lethal childhood variety of 
this disease with the adult form of the disease, 
which has been found in three siblings, and ap- 
pears to be a benign disorder. (Dr. Seegmiller 
with Dr. P. Leitman (Johns Hopkins), Dr. P. 
Frazier (NDI) and Dr. D. Shotton (Medical 
Center, Lynchburg, Va.) 

Role op Crystals op Monosodium Urate 
IN THE Genesis op Gouty Arthritis and a 
Mechanism for the Therapeutic Action of 
Colchicine. The mechanism by which crys- 
tals of monosodium urate give rise to the acute 
attack of gouty arthritis is being investigated. 
The possible role of kinin peptides in this proc- 
ess is being explored. (Dr. Seegmiller with 
Dr. Klinenberg (Johns Hopkins), and Drs. 
Melmon, Webster and Sjoerdsma (NHI).) 

GASTROENTEROLOGY UNIT 
Small Intestine 

Whipple's Intestinal Lipodystrophy. 
This study of the natural history of Whip- 
ple's disease is still in progress. One of the 



214 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



patients had been treated with antibiotics 
and steroids for only 3-4 months before her 
referral to N.I.H. in 1962, but appeared to be 
in remission as a result. She was checked at 
6-10 month intervals and did well until a re- 
lapse occurred in 1965. Studies of the morphol- 
ogy of her intestinal mucosa by light and elec- 
tron microscopy; tests of intestinal absorptive 
function ; and measurements of gastrointestinal 
protein loss are now in progress. 

The other four patients in this study .were 
treated for periods of from 2 to 3 years, and all 
appear to be doing well. One of these four was 
treated only with broad spectrum antibiotics, 
the others received the antibiotics plus ste- 
roids. (Drs. Leonard Laster, Robert Ockner, 
James Finkelstein, NIAMD; Thomas Wald- 
mann, William G. Banfield, NCI; Paul Wert- 
lake, CC.) 

Other Diseases. Studies of intestinal func- 
tion and structure and studies of protein metab- 
olism have been performed in patients with 
various diseases affecting the small bowel. The 
diseases include: scleroderma (2 patients) ; 
amyloidosis (7 patients) ; inflammatory disease 
due to ileitis and ileocolitis (2 patients) ; glu- 
ten-sensitive enteropathy (10 patients) ; lactase 
deficiency (5 patients) ; Hodgkin's disease (1 
patient) ; hypogammaglobulinemia (7 pa- 
tients) ; glycolipidosis of Fabry (2 patients); 
and vascular disease of the small intestine (2 
patients) . 

Excessive loss of protein into the gut may 
occur in almost any one of these diseases and 
when therapy is available for the underlying 
disease it appears capable of rapidly reversing 
abnormal protein exudation. The intestinal 
bacterial flora exert a profound influence on in- 
testinal function and several instances of re- 
versal of fat malabsorption by alterations of 
bacterial flora are available among these stud- 
ies. An unusual example of apparent intestinal 
lactase deficiency in identical twins, 63 years 
of age, was discovered during this work. (Drs. 
Leonard Laster, Robert Ockner, James Finkel- 
stein, NIAMD ; Thomas Waldmann, NCI; Paul 
Wertlake, CC.) 

Pathologic Physiology op the 
Intestinal Mucosa. Incubation of hamster 
small intestine in Krebs-Ringer-phosphate 
buffer containing 40 mM L-tryptophan for 20 



minutes produced a lesion in the columnar ab- 
sorptive cells characterized by apical displace- 
ment of the nuclei ; crescentic shape to nuclei ; 
extreme vacuolization of the basal half of the 
cell ; multiple projections of cytoplasm from the 
basal half of the cell ; and separation of the cell 
from, with possible disruption of, the basement 
membrane. Control studies in which the L- 
tryptophan was replaced by its D-form, gly- 
cine, or L-forms of eleven other amino acids re- 
vealed no changes similar to those produced by 
L-tryptophan. The severity of the lesion was 
proportionally related to time of exposure to 
the L-tryptophan and to the concentration of 
that amino acid. Biopsy specimens of human 
jejunal mucosa exposed to 40 mM L-trypto- 
phan for 20 minutes developed early but typi- 
cal changes of the lesion. Hamster esophagus, 
stomach, large intestine, pancreas, liver and 
kidney did not develop the typical lesion. 

Intestinal transport is disrupted by the expo- 
sure to the L-tryptophan. Whether these func- 
tional and morphological effects of L-trypto- 
phan, a natural dietary constituent, have clini- 
cal implications is under study. (Drs. Leonard 
Laster, NIAMD ; Paul Wertlake, CC) . 

Biochemistry of the Small-Intestine 
Mucosa. The in vitro incorporation of ace- 
tate-2-Ci* and mevalonate-2-C" into 27-car- 
bon sterols by scrapings of guinea pig small-in- 
testine mucosa has been investigated. About 98 
percent of the isotope incorporated into 
27-sterols was detected in lathosterol plus 7-de- 
hydrocholesterol plus cholesterol. The activity 
of mucosa from the distal two-thirds of the 
small intestine consistently exceeded that of 
the proximal small intestine. 

Preliminary studies have shown that a com- 
mercial preparation of bile salts (sodium tau- 
rocholate, 72%, sodium glycocholate, 28%) at 
4x10-^ M results in a profound inhibition of 
sterol biosynthesis from either substrate. The 
inhibition is greater with mucosa from the 
distal small intestine, the area shown by others 
to be most active in the transport of bile salts. 
(Drs. Robert Ockner and Leonard Laster, j 
NIAMD.) 

Inborn Errors op Metabolism — Homocys- 
TINURIA. The excretion of abnormal amounts 
of homocystine in the urine occurs together j 




NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



215 



with a clinical syndrome characterized by 
mental retardation, dislocated ocular lenses, 
cardiovascular disease, fatty liver, and other 
abnormalities. A patient with this syndrome 
was found to lack the enzyme cystathionine 
synthase in her liver and deficiency of this en- 
zymatic activity appears to be the fundamental 
defect in the disease. (Drs. Harvey Mudd, 
NIMH; James Finkelstein, Filadelfo Irreverre 
and Leonard Laster, NIAMD.) 

Additional studies have shown that this pa- 
tient's apparently normal parents have approx- 
imately 35 percent of the mean control value 
for hepatic cystathionine synthase activity. It 
has been proposed that the parents are hetero- 
zygous for the defect. The patient's cousin is 
mentally normal but excretes homocystine in 
the urine. Her hepatic cystathionine synthase 
activity was shown to be 12 percent of the 
mean control value. Although her genetic 
status is not clear, she demonstrates that cys- 
tathionine synthase deficiency severe enough to 
cause homocystinuria does not necessarily 
cause mental retardation. (Drs. James Finkel- 
stein, NIAMD; Harvey Mudd, NIMH; Fila- 
delfo Irreverre and Leonard Laster, NIAMD.) 

Subsequent studies have revealed that the 
patients with markedly subnormal hepatic 
cystathionine synthase activity have an im- 
paired ability to convert the sulfur of L-methi- 
onine to inorganic sulfate. This finding indi- 
cates that the defect which was discovered in 
the liver represents a generalized disturbance 
of the body's methionine metabolism ; and that 
cystathionine synthesis is an integral step in 
the major pathway for methionine catabolism 
in man. This study also revealed that during 
administration of L-methionine, the patient 
with cystathionine synthase deficiency excreted 
abnormal amounts of sulfur-containing com- 
pounds other than homocystine. The amounts 
of these urinary metabolites exceed that of ho- 
mocystine. Their identification is in progress. 
(Drs. Leonard Laster, NIAMD ; Harvey Mudd, 
NIMH ; James Finkelstein, and Filadelfo Irrev- 
erre, NIAMD.) 

CLINICAL ENDOCRINOLOGY BRANCH 

The varied activities of the Branch have con- 
tinued along similar lines as in past years, with 



the chief exception of new investigations em- 
ploying immunoassay of protein hormones. 
One member of the Branch has been assigned 
during this year to the Weizmann Institute in 
Israel, and one returned early in the year from 
the National Institute for Medical Research in 
England. The Branch has again had the benefit 
of being host to several visiting workers from 
abroad: from Belgium, England, Italy (2), 
Scotland, South Africa, and Sweden. 

Thyroid Biochemistry 

Iodide Transport. Although many sub- 
stances interfere with thyroidal iodide trans- 
port, only TSH has been known to enhance 
this transport. It has now been found that ad- 
ministration of cysteamine or cystamine to 
rats rapidly produces a 2-3 fold increase in io- 
dide accumulation when organification is 
blocked. This effect is not mediated by the kid- 
ney or the pituitary, nor by a basic change in 
the iodide concentrating mechanism, since the 
Km is unaffected. The effect may be produced 
through the Na++K+-requiring, ouabain-sensi- 
tive ATPase activity, since this system can be 
stimulated by cystamine. A secondary anti-or- 
ganification effect of these compounds (—'1/50 
the activity of methylmercaptoimidazole) ap- 
pears to be unrelated to the effect on iodide 
transport (Drs. Wolff and Rail.) 

Isolated thyroid epithelial cells, produced by 
collagenase treatment, have been found to ac- 
cumulate and organify iodide, and to possess 
an active ouabain-sensitive ATPase. Attempts 
to isolate the cell membi'anes in order to lo- 
calize both the iodide-carrying lipid and the 
ATPase in the cell have thus far been unsuc- 
cessful. (Drs. Wolff and Alexander.) 

In in vivo studies, the goitrogenic potency of 
various antithyroid anions has been predictable 
within an order of magnitude by their Ki for 
iodide transport measured in vitro. However, 
goiters produced by the anions are smaller 
than those produced by propylthiouracil in 
doses equally potent in blocking organic iodine 
formation. Furthermore, the size of the goiter 
produced by propylthiouracil is smaller when 
the anions are administered concomitantly. The 
mechanism of this "antigoitrogenic" property 



216 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of anti-thyroid anions is under investigation. 
(Drs. Wolff and Alexander.) 

lODiNATiON Reactions. In further studies 
on the chemistry of iodination of tyrosine de- 
rivatives, neutral salt had no effect on the reac- 
tion rate. Since this should affect a reaction 
between two charged species, and since evi- 
dence is strong that the phenoxide is one of the 
reactants, it is likely that the iodinating species 
is I-,. Blocking the amino and carboxyl groups 
decreases the reaction rate. Compared to tyro- 
sine, iodination of the methyl ester and the N- 
acetyl derivative are about 25% slower at pH 
9.6 in carbonate buffer. An ionizable substit- 
uent, however, (e.g., glycyl-L-tyrosine) may 
lead to a faster rate. 

Activation energies for tyrosine and most of 
its amino and carboxyl derivatives were about 
17 kcal mole-% but that for 3-iodo-L-tyrosine 
was 21 kcal mole^^ The entropies of activation 
showed similar differences (15 versus 22 e.u.). 
The relation of these values to the more rapid 
iodination of tyrosine previously observed is 
under consideration. (Drs. Rail, Mayberry, and 
Bemian— OMR.) 

Deiodination Reactions. In several bio- 
logical systems, deiodination of thyroxine in 
the presence of flavins is enhanced by ferrous 
ion, amino acids, and proteins. A study of this 
reaction from a purely chemical standpoint has 
shown that thyroxine is deiodinated in the pres- 
ence of FMN, light, oxygen and phosphate 
buffer, pH 7. The reaction is first order with 
respect to thyroxine and is proportional to 
light intensity, but FMN acts catalytically. 
Triiodothyronine is deiodinated similarly but 
at a slower rate. Amino acids, amines and pro- 
teins stimulated the reaction, but the use of a 
metal-free system prevented the stimulation by 
aliphatic amino acids. Since addition of biva- 
lent metal ions restored activity, it seems likely 
that chelated metal ion acts as an artificial 
peroxidase. Furthei' studies with a system con- 
taining hydrogen peroxide instead of FMN 
confirmed this idea, since deiodination was in- 
creased by addition of ferrous ion and versene 
together. Independently, these agents were 
without effect. (Drs. Rail and Reinwein.) 

A study of deiodination of thyroxine by thy- 
roid cell particles has been initiated. In the pres- 
ence of j8-mercaptoethanol at pH 7.5-8.1, deio- 



dination is virtually complete. The products are 
iodide and three unidentified compounds, pre- 
sumably iodothyronines. (Drs. Pastan and 
Almqvist.) 

Thyroxine Synthesis. In a continuation 
of model organic syntheses related to thyroxine 
formation, a number of new thyroxine analogs 
have been produced. Reaction of the iodinated 
metatyrosines I and II 



HO- 



> 



I, E=H 
II, E=I 



R CH2CH(NHC0CHs)C0jH 



with 3,5-diiodophenylpyruvic acid (DIHPPA), 
followed by deacetylation, gave tetra- and pen- 
taiodinated analogs III and IV. 



HO- 



R3 

1 


H, 


III, Ei=H; Rj, R3=I 


\ / 


^_0_^ ^R, 


IV, R., Rj, R3=I 

V, Ri, R2, R3=H 

VI, Ri, R3=H; R2=I 


1 


Ri CH2CH(NHs)C02H 


VII, Ri, R2=I; Rs=H 



The poor yield from this synthesis was im- 
proved by a different approach involving ether- 
ification of the ethyl esters of I and II with di- 
p-methoxy-phenyliodonium bromide. Analogs 
V, VI and VII were also synthesized. The abil- 
ity of these analogs (except V) to produce mi- 
tochondrial swelling was tested by Prof. R. 
Michel (College de France), who found that 
analogs III and IV had a behavior very like that 
of thyroxine. (Drs. Cahnmann, Shiba, and 
Hofer.) 

Further studies on the reaction of DIHPPA 
with monoiodotyrosine (MIT) and diiodotyro- 
sine (DIT) residues of thyroglobulin (TG) 
were carried out. The products differ from 
those of natural iodothyronine biosynthesis in 
that thyroxine and 3,3',5'-triiodothyronine 
(Ts') are formed in approximately equal 
amounts in the model reaction, whereas thy- 
roxine is preferentially formed in vivo. Labeled 
thyroxine and T3' could be detected only if 
slightly iodinated TG-"^I was used. This sug- 
gests that DIHPPA reacts preferentially with 
residues of MIT and DIT located at the surface 
of the protein, the latter being the major prod- 
ucts of slight iodination in vitro with ^-'^ICl. 
(Drs. Cahnmann, Toi, and Salvatore.) 

Studies on thyroxine formation in synthetic 
copolymers with polytyrosine regions have 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



217 



been initiated. These copolymers have a "back- 
bone" of poly-L-lysine, and multiple side chains 
comprised of L-tyrosine, L-glutamic acid, and 
D, L-alanine, arranged either as (l)-(ala)n- 
(glu)„-(tyr)n or (2)-(tyr),-(ala)n-(glu)n. Al- 
though the pK of the phenolic hydroxyl group 
is lower in (1), the rates of iodination, with 
ICl, of the tyrosines in (1) and (2) are similar. 
With a 20% excess of ICl, iodination of (2) is 
slightly less complete. Reaction of the partially 
iodinated polymers with DIHPPA is now 
under study. (Drs. Cahnmann and Sela — ^Weiz- 
mann Institute of Science, Israel.) 

The site of tyrosine iodination and thyroxine 
formation (either by iodination or by coupling 
with DIHPPA) in proteins of known structure 
is being investigated in lysozyme, insulin and 
ribonuclease. In lysozyme, even the tyrosine 
with pK = 12.8 is readily iodinated, and iodina- 
tion makes this residue more accessible to ti- 
tration. Thyroxine is produced under special 
circumstances not yet fully elucidated. In insu- 
lin, thyroxine is produced only (or mainly) in 
the A chain. (Drs. Wolff and Covelli.) 

lODOPROTEINS. The structure of the thyro- 
globulin molecule has been investigated 
further, particularly with respect to the effects 
of reduction of the 100 disulfide bridges in the 
molecule. Complete reduction leads to the for- 
mation of polypeptide chains which appear to 
be l^ the size of the native protein, and have 
a molecular weight of about 165,000. These are 
the smallest units obtained thus far without 
peptide bond splitting. Reoxidation of the com- 
pletely reduced molecule in air leads to the for- 
mation of a protein which closely resembles the 
native thyroglobulin molecule in sedimentation 
and viscometric properties, as well as by opti- 
cal rotary and fluorescence polarization meas- 
urements. (Drs. Edelhoch and de Crom- 
brugghe.) 

The influence of structure on the iodination 
of thyroglobulin has been explored. By iodinat- 
ing the protein in water at pH 7 or in the re- 
duced or non-reduced state in 5M guanidine or 
9M urea, it has been found that the structure 
of the protein is an important determinant of 
the resulting iodoamino acid distribution. 
(Drs. Edelhoch and Van Zyl.) 

Studies have continued on the heavy thyroi- 
dal iodoprotein, the isolation of which was de- 



scribed in last year's report. Examination of its 
physical properties revealed it to be a compact 
molecule with a sedimentation coefficient 
(S°2o,w) of 27S and a molecular weight of 
1,220,000. It had the same amino acid composi- 
tion and nitrogen content as thyroglobulin 
(19S) and had identical immunochemical de- 
terminants, but the iodine and thyroxine con- 
tent were higher than that of thyroglobulin in 
most (but not all) cases. On exposure to low 
ionic strength, alkaline pH and heat, the 27S 
protein was shown to be less stable than thyro- 
globulin and dissociated into 6S, 12S and 19S 
units. Under mild conditions, however, the 19S 
unit was the major product. The 27S protein is 
probably a dimer of thyroglobulin with a some- 
what different subunit structure. (Drs. Rob- 
bins, Cahnmann, Salvatore, and Vecchio.) 

Pulse labeling of the thyroid gland with 
^^^I- injected shortly before sacrifice was 
done in rats whose glands had been equilibrium 
labeled with "^I" by feeding the latter isotope 
for 3 or more weeks. The ratio of i^iJ/i^bx 
was much lower in the 27S protein than in thy- 
roglobulin, indicating a slower rate of iodina- 
tion of the heavy protein. These studies also 
revealed ultracentrifugal heterogeneity of thy- 
roglobulin with respect to the two isotopes, in- 
dicating a difference betAveen "young" and 
"old" thyroglobulin molecules. (Drs. Robbins, 
Salvatore, and Vecchio.) 

lodoproteins were analyzed in the blood and 
thyroid gland of South African cattle bearing 
congenital goiter. The animals were given ^-^I 
in South Africa, and the frozen materials were 
shipped to Bethesda. In the blood, the iodopro- 
tein was shoAvn by physical and immunochemi- 
cal properties to be identical with serum albu- 
min. The serum protein, as in other types of 
thyroid disease, had been iodinated in the thy- 
roid gland and released into the circulation in 
large quantity. The iodoprotein in the goiter, 
however, was very different. It was similar to, 
but not identical with, thyroglobulin by salting 
out and electrophoretic analysis, but had a 
much lower sedimentation rate ('-'9S). It did 
not form a precipitate with either antisera to 
bovine serum or to bovine thyroglobulin, but it 
did form a soluble complex with anti-bovine 
thyroglobulin. This appeared to be the first 



218 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



demonstration of an abnormal thyroglobulin in 
congenital goiter. (Drs. Robbins and Van Zyl.) 

Work has continued on the abnormal soluble 
iodoproteins found in the tumors and blood of 
rats bearing transplantable thyroid tumors 
(Dr. S. Wollman, NCI). In some timior lines, 
both tumor and blood contained iodoproteins 
with the immunochemical properties of serum 
albumin. In others, however, most of the tumor 
iodoprotein did not precipitate with anti-rat 
serum. Further studies are planned to deter- 
mine whether these tumors produce abnormal 
thyroglobulin as in the congenital goiter of 
cattle (Dr. Robbins) . 

Proteolytic Enzymes in the Thyroid 
Gland. A study has been initiated to discover 
and characterize the proteolytic systems in the 
thyroid gland which are responsible for thyro- 
globulin breakdown yielding the hormones, 
thyroxine and triiodothyronine. Previously 
described acid proteases are probably not the 
enzymes responsible for hormone secretion. A 
particulate thyroid cell fraction, sedimenting 
between 600 G and 24,000 G, hydrolyzes thyro- 
globulin over the pH range 7.0 to 10.0. Maxi- 
mum rates are obtained in the presence of 0.5 
to l.OM salt, which solubilizes the enzyme, and 
0.05M ;8-mercaptoethanol, which reduces the 
thyroglobulin, making it a better substrate. 
The products range from free iodoamino acids 
to peptides with molecular weight of about 
100,000. The iodoamino acid yield is increased 
by a heat-labile supernatant factor, probably a 
peptidase. After solubilization from cell par- 
ticles with IM KCl, the protease has been 
purified over 200 fold by gel filtration on Sepha- 
dex (5-100 and DEAE-cellulose chromatog- 
raphy. (Drs. Pastan and Almqvist.) 

Measurement of Iodocompounds in Bio- 
logical Fluids. Studies have continued on 
methods for chromatographic resolution of 
iodide and iodoamino acids on columns of ion 
exchange resins. Thyroxine and triiodothyro- 
nine have been separated using anion exchange 
resins and organic solvents of high dielectric 
constant buffered by aqueous solutions of vola- 
tile buffers. The pK's of the phenolic hydroxyl 
groups of thyroxine and triiodothyronine in 
these solvents were higher than in water, but 
the ratio of the pK's was approximately the 



same. This indicated that differential solvation 
was important in the separation. The column 
chromatography procedure is effective in sepa- 
rating milligram quantities of iodide, iodotyro- 
sines and iodothyronines, and can be used with 
thyroid gland hydrolysates. It has not yet been 
possible, however, to clarify completely the in- 
terference produced by serum. This appears to 
be due to the thyroxine-binding proteins, and 
work is currently in progress on this aspect of 
the problem. (Dr. Lewallen.) 

Thyroxine Transport in Blood. Work 
has continued on the application of the 
fluorescence quenching method to the study 
of thyroid hormone-protein interaction. Re- 
sults reported last year on the binding of 
thyroxine to serum albumin have been ex- 
tended to triiodothyronine (T3). As in the case 
of thyroxine, T3 is bound to a single strong site 
and several weaker sites. The association con- 
stant, however, is weaker in the case of T3. 
(For human serum albumin, Kt4=1.6X10" 
moles liter-i and Kt3 = 3.8x10= moles 
liter-^). These values have been confirmed 
by standard methods of equilibrium dialysis. 
(Drs. Robbins, Roth, and Steiner — Naval Med- 
ical Research Institute.) 

Anterior Pituitary Hormones 

Growth Hormone. The properties of bo- 
vine growth hormone have been studied by 
optical rotation, sedimentation, viscosity, fluo- 
rescence and fluorescence polarization. A molecu- 
lar transition occurs in acid (pK=3.55) and 
also in the neutral pH range. These molecular 
changes are reversible if ionic strength is 
maintained at a low level. Further configura- 
tional changes have been observed in urea. 
(Drs. Edelhoch, Condliflfe (LNE), and Burger 
(LNE).) 

A specific immunoassay for human growth 
hormone (HGH) has recently been developed 
and used to define the plasma HGH level in 
man and its response to changes in blood glu- 
cose. It has already been found that plasma 
HGH is elevated in acromegaly. The present 
studies, in which 25 patients have thus far 
been included, is planned to correlate the ab- 
normalities in HGH function with the usual 
clinical measurements, to define criteria for 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



219 



diagnosis, and to determine the response of 
plasma HGH to pituitary irradiation and estro- 
gen therapy. (Drs. Roth and Click — Maimon- 
edes Hospital.) 

Although prolactin and growth hormone are 
separate hormones in non-primates, attempts 
to separate them in primates have met with 
little success. Plasma samples have been as- 
sayed for HGH by radioimmunoassay and for 
prolactin by pigeon crop sac assay. In acrome- 
galics, plasma HGH is high but prolactin is 
normal. In women 48 hours postpartum, HGH 
is normal but prolactin is elevated. (Drs. Roth 
and Bates.) 

Presently available assay methods for 
plasma growth hormone are limited to pri- 
mates. In order to enable future studies on 
growth hormone physiology in animals, work is 
under way to prepare rat gro'wth hormone 
from over 1000 rat pituitary glands. Antisera 
which react strongly with monkey growth hor- 
mone are also being developed. (Dr. Roth.) 

Gonadotropins. Studies on gynecomastia 
in bronchogenic carcinoma have continued. 
Three men with this syndrome excreted 250 to 
50,000 times as much gonadotropic activity in 
their urine as did eleven control men with 
bronchogenic carcinoma but no gsmecomastia. 
In two cases available at autopsy, the pituitary 
gland was depleted of gonadotropin, whereas 
the tumor contained high titers of gonadotrop- 
ic activity. Apparently these tumors produced 
a gonadotropin-like substance which was re- 
sponsible for the gynecomastia. (Drs. Rosen 
and Fusco (Mt. Alto V.A. Hospital).) 

Further studies have been carried out on the 
syndromes of hypogonadism and anosmia. At 
least two distinct syndromes have been recog- 
nized. Five patients with the classic syndrome 
showed decreased urinary gonadotropin 
(mouse uterus) and no gametogenesis. Patients 
with variants of the syndrome may excrete 
normal amounts of urinary gonadotropin, may 
have gametogenesis, and may have midline 
cranial defects (cleft lip, etc.). In a kindred of 
165 members with a high rate of consanguin- 
ity, five cases with hypogonadism, three with 
anosmia, and six with midline anomalies have 
been found. Four cases had more than one of 
these abnormalities. The pedigree was com- 



patible with autosomal recessive inheritance. 
(Dr. Rosen.) 

A crude preparation of human pituitary 
FSH has been further purified by gel filtration 
on Sephadex G-lOO and DEAE-cellulose chro- 
matogi'aphy. Fractions with 90% or more of 
the protein in a single band on acrylamide gel 
electrophoresis have been obtained. Work is 
under way to obtain antibodies to FSH and to 
iodinate the purified protein in order to develop 
an immunoassay for FSH. (Drs. Rosen, 
Schlaff. and Roth.) 

Thyrotropin. A small quantity of highly 
purified human thyroid stimulating hormone 
(TSH) was available for study. This material 
was assayed in 7 human subjects and compared 
with bovine TSH. In one subject a dose re- 
sponse curve was obtained. The response was 
measured by performing 2- or 3-hour thyroid 
uptakes of "^I, which could be given in mul- 
tiple doses. No significant difference was found 
between human and bovine TSH either in the 
time-course of action or in the dose-response. 
TSH does not, therefore, share the striking- 
species specificity of growth hormone, and pre- 
vious estimates of human TSH secretion rate 
based on response to bovine TSH are con- 
firmed. (Drs. Robbins, Schneider, and Condliffe 
— LNE.) 

Steroid Hormones 

Since the last report, the survey of the 
synthesis of steroid sulfates by the soluble 
fraction of various bovine tissues has been 
completed. All tissues synthesized p-nitrophe- 
nol sulfate. Testosterone sulfate was formed 
only by liver. Estrone sulfate was formed by 
many tissues, and dehydroepiandrosterone sul- 
fate was formed by placenta, liver, adrenal, in- 
testine, pituitary, kidney and uterus. Bovine 
placenta was much more active than human 
placenta in the sulfation reaction. 

The enzymes which catalyze the transfer of 
sulfate from adenosine-3'-phosphate-5'-phos- 
phosulfate to hydroxylated steroids are now 
being purified. There appears to be a family of 
enzymes specific for the various hydroxy] 
groups on the steroid nucleus. (Drs. Rosen and 
Holcenberg.) 



220 



ANNUAL REVIEW OF INTKAMURAL RESEARCH 



Insulin, Glucagon and Carbohydrate Metabo- 
lism 

Action of Insulin on the Liver. The 
inhibition of net protein degradation in the iso- 
lated, cyclically perfused rat liver which is pro- 
duced by insulin in vitro has received further 
study. L-valine-C" was used, since this amino 
acid is presumably not synthesized and only 
negligible amounts are metabolized in the per- 
fused liver. The steady increase of free valine 
in the perfusate as a function of time was in- 
hibited 65% by insulin, and valine-C" release 
from previously labeled liver was also inhibit- 
ed. On the other hand, rates of incorporation of 
valine into liver and serum protein were unaf- 
fected by insulin, nor was there an effect on 
valine distribution between intra- and extracel- 
lular pools nor on release of labeled protein. 
These findings strongly support the view that 
insulin acts at a site which normally limits the 
rate of protein degradation. Specific inductive 
effects on specific enzjones, however, are not 
excluded. (Drs. Mortimore and Mondon.) 

Action of Glucagon on the Liver. Glu- 
cagon is known to enhance protein catabolism 
in vivo and to stimulate urea formation in the 
isolated liver. Studies have been initiated using 
the cyclically perfused liver in order to identify 
the mechanism of these effects. In the perfused 
liver, glucagon markedly enhances urea forma- 
tion but has no effect on total amino acid accu- 
mulation. Chromatographic analysis of perfu- 
sate amino acids, however, revealed a striking 
alteration. Glucagon caused a 30 to 40% in- 
crease in valine, lysine, leucine and isoleucine, 
and a 50 to 80% decrease in alanine, gluta- 
mate, glutamine and several other amino acids. 
The urea effect could also be produced by epin- 
ephrine and 3',5'-cyclic AMP, but not by 5'- 
AMP or ATP. The possibility of two effects of 
glucagon — one on amino acid metabolism per 
se, and another on net protein degradation — is 
under investigation. (Drs. Mortimore and 
Mondon.) 

Glucose Metabolism, Diabetes and Hypo- 
glycemia. Work is continuing on the kinet- 
ic analysis of ^*C-glucose metabolism in man. 
Utilizing the previously described model, the 
kinetics of C-6 labeled glucose from the time 
of its disappearance from blood until its appear- 



ance as "CO2 has been analyzed. Two expo- 
nentials have been derived for the characteriza- 
tion of the whole body Emden-Meyerhoff path- 
way. (Drs. Segal, H. Roth, and Berman — 
OMR.) 

Previous studies had demonstrated that glu- 
cose inhibits amino acid transport in kidney 
cortex slices. A study of aminoaciduria in dia- 
betes has, therefore, been initiated. In the 
small number of patients thus far studied, 
some had generalized aminoaciduria and one 
excreted only cystine and lysine. (Drs. Segal 
and Schwartzman.) 

Observations have continued on the use of 
long acting zinc glucagon in the management 
of hypoglycemia. One patient with islet cell 
adenoma, and one with islet cell carcinoma, 
have been successfully treated for periods of 
one to two years. Combined treatment with 
zinc glucagon and adrenal steroid is being ex- 
plored. (Drs. Segal and H. Roth.) 

Galactose Metabolism and Galactose- 
mia. Studies have continued on the pos- 
sible effects of orotic acid in galactosemia. 
This substance, a precursor of cofactors impor- 
tant in galactose metabolism, had been report- 
ed by others to be effective in enhancing galac- 
tose tolerance. As reported last year, orotic 
acid had no effect on galactose-^^C metabolism 
in normal or galactosemic subjects. It has also 
been found to be without effect in protecting 
rats from galactose-induced cataracts. (Drs. 
Segal and H. Roth.) 

Further studies have been done on the na- 
ture of galactose toxicity in newborn rats pro- 
duced by galactose feeding during pregnancy. 
In addition to decreased weight of the newborn 
and of the placenta, cataracts, liver damage 
and abnormalities of renal tubule cells have 
been observed. Although histochemical studies 
indicate markedly reduced liver glycogen, this 
was not confirmed by chemical analysis. The 
possible existence of an abnormal form of liver 
glycogen in galactosemia is suggested. (Drs. 
Segal and Spatz — Lab. of Histochem.) 

It was reported last year that galactose utili- 
zation by rat liver decreases markedly with 
age. Since galactose-1-P uridyl transfera